Human binding molecules having killing activity against enterococci

ABSTRACT

Described are human binding molecules specifically binding to enterococci and having killing activity against enterococci, nucleic acid molecules encoding the human binding molecules, compositions comprising the human binding molecules and methods of identifying or producing the molecules. The molecules can be used, for example, in the diagnosis, prophylaxis, and/or treatment of a condition resulting from  Enterococcus.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of co-pending U.S. patent applicationSer. No. 13/550,100, filed Jul. 16, 2012, which application is acontinuation of U.S. patent application Ser. No. 13/068,784, filed May19, 2011, now U.S. Pat. No. 8,241,631 (issued Aug. 14, 2012), which is adivisional of U.S. patent application Ser. No. 12/227,116, filed Nov. 7,2008, now U.S. Pat. No. 7,960,518 (issued Jun. 14, 2011), which is anational phase entry of PCT International Patent Application No.PCT/EP2007/055535, filed on Jun. 5, 2007, designating the United Statesof America, and published, in English, as PCT International PublicationNo. WO 2007/141278 A2 on Dec. 13, 2007, which claims the benefit underArticle 8 of the PCT of EP 06115013.2, filed Jun. 6, 2006, EP06116719.2, filed Jul. 6, 2006, EP 06121258.5, filed on Sep. 26, 2006,and EP 07103587.7 filed on Mar. 6, 2007 and under Article 8 of the PCTand 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No.60/811,542, filed Jun. 6, 2006, the disclosures of each of which arehereby incorporated herein in its entirety by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e) Sequence ListingSubmitted as PDF File With a Request to Transfer CRF from ParentApplication

Pursuant to 37 C.F.R. §1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

The disclosure relates generally to biotechnology and medicine. Inparticular, the disclosure relates to the diagnosis, prophylaxis and/ortreatment of infection, such as by Enterococci.

BACKGROUND

Enterococci are gram-positive, facultatively anaerobic bacteria of thefamily Enterococcaceae. They were previously classified as Group Dstreptococci. Enterococci are found in the bowels of most humans and arecommonly isolated from stool, urine and sites of intra-abdominal andlower extremity infection. Bacteria of the genus Enterococcus are oftenregarded as harmless commensals of the gastrointestinal tract, butwithin the last 10 years they have become an important cause ofnosocomial (hospital-acquired) infections, not because of increasedvirulence but because of antibiotic resistance. It has been estimated inthe United States of America, that 800,000 cases of enterococcalinfection occur each year resulting in costs of around $500 million. Toinfect hosts, enterococci primarily colonize mucosal surfaces.Enterococci are etiological agents of bacteremia, surgical woundinfections, urinary tract infections, and endocarditis. They are alsoassociated with obligate anerobes in mixed infections that result inintra-abdominal abscesses. Overall, there are about seventeen species ofenterococci, among which Enterococcus faecalis and Enterococcus faeciumappear to be the most commonly detected in human feces. E. faecalisaccounts for most of the enterococcal infections of humans, usuallyrepresenting 80-90% of clinical isolates. E. faecium is detected muchless frequently but is nevertheless of significance because of a highincidence of multiple resistances to antibacterial agents. Enterococcalinfections are commonly treated with antimicrobials and until recentlythey have been adequately controlled using these agents. However,drug-resistant enterococcal strains are emerging, and infection bystrains resistant to all presently available antibiotics may become aserious problem in the near future. Some enterococci have alreadyacquired intrinsic resistance to β-lactam-based antibiotics(penicillins) as well as many aminoglycosides. In the last two decades,particularly virulent strains of Enterococcus which are even resistantto the antibiotic vancomycin (Vancomycin-Resistant Enterococcus, or VRE)have emerged in nosocomial infections of hospitalized patients. Despitethe urgent need for the development of new antibiotics, the majorpharmaceutical companies appear to have lost interest in the antibioticmarket. In 2002, only 5 out of the more than 500 drugs in phase II orphase III clinical development were new antibiotics. In the last 6 yearsonly 10 antibiotics have been registered and only 2 of those did notexhibit cross-reactivity with existing drugs (and thus not subject tothe same patterns of drug resistance). This trend has been attributed toseveral factors: the cost of new drug development and the relativelysmall return on investment that infectious disease treatments yieldcompared to drugs against hypertension, arthritis and lifestyle drugs,e.g., for impotence. Another contributing factor is the increasingdifficulty in finding new targets, further driving up development costs.Therefore, investigation into novel therapies or preventative measuresfor (multi-drug-resistant) bacterial infections is urgently needed tomeet this impending healthcare crisis.

Active immunization with vaccines and passive immunization withimmunoglobulins are promising alternatives to classical small moleculetherapy. A few bacterial diseases that once caused widespread illness,disability, and death can now be prevented through the use of vaccines.The vaccines are based on weakened (attenuated) or dead bacteria,components of the bacterial surface or on inactivated toxins. The immuneresponse raised by a vaccine is mainly directed to immunogenicstructures, a limited number of proteins or sugar structures on thebacteria that are actively processed by the immune system. Since theseimmunogenic structures are very specific to the organism, the vaccineneeds to comprise the immunogenic components of all variants of thebacteria against which the vaccine should be protective. As aconsequence thereof, vaccines are very complex, take long and areexpensive to develop. Further complicating the design of vaccines is thephenomenon of “antigen replacement.” This occurs when new strains becomeprevalent that are serologically and thus antigenically distinct fromthose strains covered by the vaccines. The immune status of thepopulations at risk for nosocomial infections further complicatesvaccine design. These patients are inherently unwell and may even beimmunocompromised (due to the effect of immunosuppressive drugs)resulting in delayed or insufficient immunity against the infectingpathogens. Furthermore, except in the case of certain electiveprocedures, it may not be possible to identify and vaccinate the at riskpatients in time to give them sufficient immune protection frominfection.

Direct administration of therapeutic immunoglobulins, also referred toas passive immunization, does not require an immune response from thepatient and therefore gives immediate protection. In addition, passiveimmunization can be directed to bacterial structures that are notimmunogenic and that are less specific to the organism. Passiveimmunization against pathogenic organisms has been based onimmunoglobulins derived from sera of human or non-human donors. However,blood-derived products have potential health risks inherently associatedwith these products. In addition, the immunoglobulins can displaybatch-to-batch variation and may be of limited availability in case ofsudden mass exposures. Recombinantly produced antibodies do not havethese disadvantages and thus offer an opportunity to replaceimmunoglobulins derived from sera.

Murine monoclonal antibodies directed against enterococcal antigens areknown in the art (see, WO 03/072607). However, murine antibodies arelimited for their use in vivo due to problems associated withadministration of murine antibodies to humans, such as short serum halflife, an inability to trigger certain human effector functions andelicitation of an unwanted dramatic immune response against the murineantibody in a human (HAMA).

WO 99/18996 relates to enterococcus antigens and vaccines. WO 99/18996further discloses rabbit antiserum against conjugated purified antigensfrom enterococci, and opsonic activity of such antiserum.

Although WO 99/18996 refers to human antibodies as desired molecules,the antibodies actually disclosed and used therein are of rabbit origin,and this document actually does not actually disclose any humanantibodies, and does not disclose sequences thereof.

SUMMARY

Disclosed are human binding molecules able to specifically bind toenterococci and exhibiting killing and/or growth inhibiting activityagainst enterococci. The disclosure also pertains to nucleic acidmolecules encoding at least the binding region of these human bindingmolecules. Further provided for is the use of the human bindingmolecules hereof in the prophylaxis and/or treatment of a subjecthaving, or at the risk of developing, an Enterococcus infection. Besidesthat, the disclosure pertains to the use of the human binding moleculesin the diagnosis/detection of Enterococcus.

DESCRIPTION OF THE FIGURES

FIG. 1 shows data of an in vivo experiment. On the Y-axis CFU/ml inblood of mice is shown, while on the X-axis the respective antibodiesare depicted. The antibodies were used in an amount of 15 mg/kg, withthe exception of CR6016 and CR6241 which were used in an amount of 7.5mg/kg. With the exception of CR6043 and CR6071 all of the antibodies hada median that differed significantly with that of the control IgG(P<0.05 vs. IgG1 Ctrl.).

DETAILED DESCRIPTION

The term “amino acid sequence,” as used herein, refers to naturallyoccurring or synthetic molecules and to a peptide, oligopeptide,polypeptide, or protein sequence.

As used herein, the term “binding molecule” refers to an intactimmunoglobulin including monoclonal antibodies, such as chimeric,humanized or human monoclonal antibodies, or to an antigen-bindingand/or variable domain comprising fragment of an immunoglobulin thatcompetes with the intact immunoglobulin for specific binding to thebinding partner of the immunoglobulin, e.g., enterococci. Regardless ofstructure, the antigen-binding fragment binds with the same antigen thatis recognized by the intact immunoglobulin. An antigen-binding fragmentcan comprise a peptide or polypeptide comprising an amino acid sequenceof at least 2 contiguous amino acid residues, at least 5 contiguousamino acid residues, at least 10 contiguous amino acid residues, atleast 15 contiguous amino acid residues, at least 20 contiguous aminoacid residues, at least 25 contiguous amino acid residues, at least 30contiguous amino acid residues, at least 35 contiguous amino acidresidues, at least 40 contiguous amino acid residues, at least 50contiguous amino acid residues, at least 60 contiguous amino residues,at least 70 contiguous amino acid residues, at least 80 contiguous aminoacid residues, at least 90 contiguous amino acid residues, at least 100contiguous amino acid residues, at least 125 contiguous amino acidresidues, at least 150 contiguous amino acid residues, at least 175contiguous amino acid residues, at least 200 contiguous amino acidresidues, or at least 250 contiguous amino acid residues of the aminoacid sequence of the binding molecule.

The term “binding molecule,” as used herein, includes all immunoglobulinclasses and subclasses known in the art. Depending on the amino acidsequence of the constant domain of their heavy chains, binding moleculescan be divided into the five major classes of intact antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.

Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv,dAb, Fd, complementarity-determining region (CDR) fragments,single-chain antibodies (scFv), bivalent single-chain antibodies,single-chain phage antibodies, diabodies, triabodies, tetrabodies,peptides or polypeptides that contain at least a fragment of animmunoglobulin that is sufficient to confer specific antigen binding tothe peptide or polypeptide, etc. The above fragments may be producedsynthetically or by enzymatic or chemical cleavage of intactimmunoglobulins or they may be genetically engineered by recombinant DNAtechniques. The methods of production are well-known in the art and aredescribed, for example, in Antibodies: A Laboratory Manual, Edited by:E. Harlow and D, Lane (1988), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., which is incorporated herein by reference. A bindingmolecule or antigen-binding fragment thereof may have one or morebinding sites. If there is more than one binding site, the binding sitesmay be identical to one another or they may be different.

The binding molecule can be a naked or unconjugated binding molecule,but can also be part of an immunoconjugate. A naked or unconjugatedbinding molecule is intended to refer to a binding molecule that is notconjugated, operatively linked or otherwise physically or functionallyassociated with an effector moiety or tag, such as inter alia a toxicsubstance, a radioactive substance, a liposome, an enzyme. It will beunderstood that naked or unconjugated binding molecules do not excludebinding molecules that have been stabilized, multimerized, humanized orin any other way manipulated, other than by the attachment of aneffector moiety or tag. Accordingly, all post-translationally modifiednaked and unconjugated binding molecules are included herewith,including where the modifications are made in the natural bindingmolecule-producing cell environment, by a recombinant bindingmolecule-producing cell, and are introduced by the hand of man afterinitial binding molecule preparation. The term naked or unconjugatedbinding molecule does not exclude the ability of the binding molecule toform functional associations with effector cells and/or molecules afteradministration to the body, as some of such interactions are necessaryin order to exert a biological effect. The lack of associated effectorgroup or tag is therefore applied in definition to the naked orunconjugated binding molecule in vitro, not in vivo.

As used herein, the term “biological sample” encompasses a variety ofsample types, including blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures, or cells derived therefrom and the progeny thereof. The termalso includes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides.The term encompasses various kinds of clinical samples obtained from anyspecies, and also includes cells in culture, cell supernatants and celllysates.

The term “complementarity-determining regions” (CDR) as used hereinmeans sequences within the variable regions of binding molecules, suchas immunoglobulins, that usually contribute to a large extent to theantigen binding site which is complementary in shape and chargedistribution to the epitope recognized on the antigen. The CDR regionscan be specific for linear epitopes, discontinuous epitopes, orconformational epitopes of proteins or protein fragments, either aspresent on the protein in its native conformation or, in some cases, aspresent on the proteins as denatured, e.g., by solubilization in SDS.Epitopes may also consist of posttranslational modifications ofproteins.

The term “deletion,” as used herein, denotes a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent as compared to the parent,often the naturally occurring, molecule.

The term “expression-regulating nucleic acid sequence,” as used herein,refers to polynucleotide sequences necessary for and/or affecting theexpression of an operably linked coding sequence in a particular hostorganism. The expression-regulating nucleic acid sequences, such asinter alia appropriate transcription initiation, termination, promoter,enhancer sequences; repressor or activator sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (e.g., ribosome binding sites); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion, can be any nucleic acid sequence showing activity inthe host organism of choice and can be derived from genes encodingproteins, which are either homologous or heterologous to the hostorganism. The identification and employment of expression-regulatingsequences is routine to the person skilled in the art.

The term “functional variant,” as used herein, refers to a bindingmolecule that comprises a nucleotide and/or amino acid sequence that isaltered by one or more nucleotides and/or amino acids compared to thenucleotide and/or amino acid sequences of the parental binding moleculeand that is still capable of competing for binding to the bindingpartner, e.g., enterococci, with the parental binding molecule. In otherwords, the modifications in the amino acid and/or nucleotide sequence ofthe parental binding molecule do not significantly affect or alter thebinding characteristics of the binding molecule encoded by thenucleotide sequence or containing the amino acid sequence, i.e., thebinding molecule is still able to recognize and bind its target. Thefunctional variant may have conservative sequence modificationsincluding nucleotide and amino acid substitutions, additions anddeletions. These modifications can be introduced by standard techniquesknown in the art, such as site-directed mutagenesis and randomPCR-mediated mutagenesis, and may comprise natural as well asnon-natural nucleotides and amino acids.

Conservative amino acid substitutions include the ones in which theamino acid residue is replaced with an amino acid residue having similarstructural or chemical properties. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.,glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan). Other classifications of amino acid residue families thanthe one used hereinbefore can also be employed. Furthermore, a variantmay have non-conservative amino acid substitutions, e.g., replacement ofan amino acid with an amino acid residue having different structural orchemical properties. Similar minor variations may also include aminoacid deletions or insertions, or both. Guidance in determining whichamino acid residues may be substituted, inserted, or deleted withoutabolishing immunological activity may be found using computer programswell-known in the art.

A mutation in a nucleotide sequence can be a single alteration made at alocus (a “point mutation”), such as transition or transversionmutations, or alternatively, multiple nucleotides may be inserted,deleted or changed at a single locus. In addition, one or morealterations may be made at any number of loci within a nucleotidesequence. The mutations may be performed by any suitable method known inthe art.

The term “host,” as used herein, is intended to refer to an organism ora cell into which a vector such as a cloning vector or an expressionvector has been introduced. The organism or cell can be prokaryotic oreukaryotic. It should be understood that this term is intended to refernot only to the particular subject organism or cell, but to the progenyof such an organism or cell as well. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentorganism or cell, but are still included within the scope of the term“host” as used herein.

The term “human,” when applied to binding molecules as defined herein,refers to molecules that are either directly derived from a human orbased upon a human sequence. When a binding molecule is derived from orbased on a human sequence and subsequently modified, it is still to beconsidered human as used throughout the specification. In other words,the term human, when applied to binding molecules is intended to includebinding molecules having variable and constant regions derived fromhuman germline immunoglobulin sequences or based on variable or constantregions occurring in a human or human lymphocyte and modified in someform. Thus, the human binding molecules may include amino acid residuesnot encoded by human germline immunoglobulin sequences; comprisesubstitutions and/or deletions (e.g., mutations introduced by forinstance random or site-specific mutagenesis in vitro or by somaticmutation in vivo). “Based on,” as used herein, refers to the situationthat a nucleic acid sequence may be exactly copied from a template, orwith minor mutations, such as by error-prone PCR methods, orsynthetically made matching the template exactly or with minormodifications. Semi-synthetic molecules based on human sequences arealso considered to be human as used herein.

The term “insertion,” also known as the term “addition,” denotes achange in an amino acid or nucleotide sequence resulting in the additionof one or more amino acid or nucleotide residues, respectively, ascompared to the parent sequence.

The term “intrinsic activity,” when applied to binding molecules asdefined herein, refers to binding molecules that are capable of bindingto certain protein or carbohydrate antigens on the surface of pathogenssuch as bacteria and that can inhibit the ability of the pathogen togrow and divide normally. Such binding molecules can for example blockthe entry of specific nutrients required for growth or the transport oftoxic waste elements from the bacteria. Through the latter action theymay also increase the sensitivity of bacteria to the action ofantibiotic drugs.

The term “isolated,” when applied to binding molecules as definedherein, refers to binding molecules that are substantially free of otherproteins or polypeptides, particularly free of other binding moleculeshaving different antigenic specificities, and are also substantiallyfree of other cellular material and/or chemicals. For example, when thebinding molecules are recombinantly produced, they are preferablysubstantially free of culture medium, and when the binding molecules areproduced by chemical synthesis, they are preferably substantially freeof chemical precursors or other chemicals, i.e., they are separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. The term “isolated” when applied to nucleicacid molecules encoding binding molecules as defined herein, is intendedto refer to nucleic acid molecules in which the nucleotide sequencesencoding the binding molecules are free of other nucleotide sequences,particularly nucleotide sequences encoding binding molecules that bindbinding partners other than enterococci. Furthermore, the term“isolated” refers to nucleic acid molecules that are substantiallyseparated from other cellular components that naturally accompany thenative nucleic acid molecule in its natural host, e.g., ribosomes,polymerases, or genomic sequences with which it is naturally associated.Moreover, “isolated” nucleic acid molecules, such as cDNA molecules, canbe substantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The term “monoclonal antibody,” as used herein, refers to a preparationof antibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody displaying a single binding specificity which hasvariable and constant regions derived from or based on human germlineimmunoglobulin sequences or derived from completely synthetic sequences.The method of preparing the monoclonal antibody is not relevant.

The term “naturally occurring,” as used herein, as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organismthat can be isolated from a source in nature and which has not beenintentionally modified by man in the laboratory is naturally occurring.

The term “nucleic acid molecule,” as used herein, refers to a polymericform of nucleotides and includes both sense and anti-sense strands ofRNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of theabove. A nucleotide refers to a ribonucleotide, deoxynucleotide or amodified form of either type of nucleotide. The term also includessingle- and double-stranded forms of DNA. In addition, a polynucleotidemay include either or both naturally occurring and modified nucleotideslinked together by naturally occurring and/or non-naturally occurringnucleotide linkages. The nucleic acid molecules may be modifiedchemically or biochemically or may contain non-natural or derivatizednucleotide bases, as will be readily appreciated by those of skill inthe art. Such modifications include, for example, labels, methylation,substitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates,carbamates, etc.), charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators,and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Theabove term is also intended to include any topological conformation,including single-stranded, double-stranded, partially duplexed, triplex,hairpinned, circular and padlocked conformations. Also included aresynthetic molecules that mimic polynucleotides in their ability to bindto a designated sequence via hydrogen bonding and other chemicalinteractions. Such molecules are known and include, for example, thosein which peptide linkages substitute for phosphate linkages in thebackbone of the molecule. A reference to a nucleic acid sequenceencompasses its complement unless otherwise specified. Thus, a referenceto a nucleic acid molecule having a particular sequence should beunderstood to encompass its complementary strand, with its complementarysequence. The complementary strand is also useful, e.g., for anti-sensetherapy, hybridization probes and PCR primers.

The term “operably linked” refers to two or more nucleic acid sequenceelements that are usually physically linked and are in a functionalrelationship with each other. For instance, a promoter is operablylinked to a coding sequence, if the promoter is able to initiate orregulate the transcription or expression of a coding sequence, in whichcase the coding sequence should be understood as being “under thecontrol of” the promoter.

“Opsonic activity” refers to the ability of an opsonin (generally eithera binding molecule, e.g., an antibody, or serum complement factors) tobind to the surface of a pathogen either by specific antigenicrecognition (in the case of antibodies) or through the catalytic effectof surface bound molecules (e.g., the increased deposition of C3b as aresult of surface bound antibodies). Phagocytosis of opsonized pathogensis enhanced due to the specific recognition of receptors on thephagocyte for the opsonin (the Fc receptor in case the antibodiesthemselves are the opsonins and the complement receptor in casecomplement is the opsonin). Certain bacteria, especially encapsulatedbacteria that resist phagocytosis due to the presence of the capsule,become extremely attractive to phagocytes such as neutrophils andmacrophages when coated with an opsonic antibody and their rate ofclearance from the bloodstream and infected organs is strikinglyenhanced. Opsonic activity may be measured in any conventional manner(e.g., the opsonic phagocytic killing assay).

By “pharmaceutically acceptable excipient” is meant any inert substancethat is combined with an active molecule such as a drug, agent, orbinding molecule for preparing an agreeable or convenient dosage form.The “pharmaceutically acceptable excipient” is an excipient that isnon-toxic to recipients at the dosages and a concentration employed, andis compatible with other ingredients of the formulation comprising thedrug, agent or binding molecule.

The term “specifically binding,” as used herein, in reference to theinteraction of a binding molecule, e.g., an antibody, and its bindingpartner, e.g., an antigen, means that the interaction is dependent uponthe presence of a particular structure, e.g., an antigenic determinantor epitope, on the binding partner. In other words, the antibodypreferentially binds or recognizes the binding partner even when thebinding partner is present in a mixture of other molecules or organisms.The binding may be mediated by covalent or non-covalent interactions ora combination of both. In yet other words, the term “specificallybinding” means immunospecifically binding to an antigen or a fragmentthereof and not immunospecifically binding to other antigens. A bindingmolecule that immunospecifically binds to an antigen may bind to otherpeptides or polypeptides with lower affinity as determined by, e.g.,radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA),BIACORE, or other assays known in the art. Binding molecules orfragments thereof that immunospecifically bind to an antigen may becross-reactive with related antigens. Preferably, binding molecules orfragments thereof that immunospecifically bind to an antigen do notcross-react with other antigens.

A “substitution,” as used herein, denotes the replacement of one or moreamino acids or nucleotides by different amino acids or nucleotides,respectively.

The term “therapeutically effective amount” refers to an amount of thebinding molecule effective for preventing, ameliorating and/or treatinga condition resulting from infection with Enterococcus.

The term “treatment” refers to therapeutic treatment as well asprophylactic or preventative measures to cure or halt or at least retarddisease progress. Those in need of treatment include those alreadyinflicted with a condition resulting from infection with Enterococcus aswell as those in which infection with Enterococcus is to be prevented.Subjects partially or totally recovered from infection with Enterococcusmight also be in need of treatment. Prevention encompasses inhibiting orreducing the spread of Enterococcus or inhibiting or reducing the onset,development or progression of one or more of the symptoms associatedwith infection with Enterococcus.

The term “vector” denotes a nucleic acid molecule into which a secondnucleic acid molecule can be inserted for introduction into a host whereit will be replicated, and in some cases expressed. In other words, avector is capable of transporting a nucleic acid molecule to which ithas been linked. Cloning as well as expression vectors are contemplatedby the term “vector,” as used herein. Vectors include, but are notlimited to, plasmids, cosmids, bacterial artificial chromosomes (BAC)and yeast artificial chromosomes (YAC) and vectors derived frombacteriophages or plant or animal (including human) viruses. Vectorscomprise an origin of replication recognized by the proposed host and incase of expression vectors, promoter and other regulatory regionsrecognized by the host. A vector containing a second nucleic acidmolecule is introduced into a cell by transformation, transfection, orby making use of viral entry mechanisms. Certain vectors are capable ofautonomous replication in a host into which they are introduced (e.g.,vectors having a bacterial origin of replication can replicate inbacteria). Other vectors can be integrated into the genome of a hostupon introduction into the host, and thereby are replicated along withthe host genome.

In a first aspect, disclosed are binding molecules capable ofspecifically binding to an Enterococcus species. The binding moleculesmay be human binding molecules. The binding molecules may exhibitkilling activity against an Enterococcus species. In a further aspect,the binding molecules hereof are capable of specifically binding toand/or have killing activity against at least two different Enterococcusspecies. Preferably, the binding molecules hereof are capable ofspecifically binding to and/or have killing activity against at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, at least ten, at least eleven, at leasttwelve, at least thirteen, at least fourteen, at least fifteen, at leastsixteen, at least seventeen different Enterococcus species. Enterococcusspecies that the binding molecules hereof are capable of specificallybinding to and/or have killing activity against are selected from thegroup consisting of E. asini, E. avium, E. casseliflavus, E. cecorum, E.columbae, E. dispar, E. durans, E. faecalis, E. faecium, E. flavescens,E. gallinarum, E. gilvus, E. haemoperxidus, E. hirae, E. malodoratus, E.moraviensis, E. mundtii, E. pallens, E. porcinus, E. pseudoavium, E.raffinosus, E. ratti, E. saccharolyticus, E. seriolicida, E. solitarius,E. sulfureus, E. villorum, with E. faecalis and E. faecium beingpreferred species. In an embodiment, the binding molecules hereof arecapable of specifically binding to and have killing activity againstdifferent strains within one Enterococcus species. In anotherembodiment, the binding molecules hereof may even be capable ofspecifically binding to and/or have killing activity against at leastone other Gram-positive bacterium and/or Gram-negative bacteriumincluding, but not limited to, Group A streptococci; Streptococcuspyrogenes, Group B streptococci; Streptococcus agalactiae, Streptococcusmilleri, Streptococcus pneumoniae, Viridans streptococci; Streptococcusmutans, Staphylococcus aureus, Staphylococcus epidermidis,Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacteriumpseudotuberculosis, Corynebacterium jeikeium, Corynebacterium xerosis,Corynebacterium pseudodiphtheriticum, Bacillus anthracis, Bacilluscereus, Listeria monocytogenes, Clostridium perfringens, Clostridiumtetani, Clostridium botulinum, Clostridium difficile, Mycobacteriumtuberculosis, Mycobacterium leprae, Actinomyces israelii, Norcardiaasteroides, Norcardia brasiliensis, Escherichia coli, Proteus mirabilis,Proteus vulgaris, Klebsiella pneumoniae, Salmonella typhi, Salmonellaparatyphi A, B & C, Salmonella enteritidis, Salmonella cholerae-suis,Salmonella virchow, Salmonella typhimurium, Shigella dysenteriae,Shigella boydii, Shigella flexneri, Shigella sonnei, Pseudomonasaeruginosa, Pseudomonas mallei, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Campylobacterpylori, Helicobacter pylori, Campylobacter jejuni, Bacteroides fragilis,Neisseria gonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Haemophilus influenzae, Haemophilus ducreyi, Bordetella pertussis,Brucella abortus, Brucella abortus, Brucella melitensis, Legionellapneumophila, Treponema pallidum, Treponema carateum, Leptospirainterrogans, Leptospira biflexa, Borrelia recurrentis, Borreliaburgdorferi, Mycoplasma pneumoniae, Coxiella burnetii, Clamydiatrachomatis, Clamydia psittaci, Clamydia pneumoniae. The bindingmolecules hereof may be capable of specifically binding to enterococciand optionally other Gram-positive and/or Gram-negative bacteria thatare viable, living and/or infective or that are ininactivated/attenuated form. Methods for inactivating/attenuatingbacteria are well-known in the art and include, but are not limited to,antibiotic treatment, UV treatment, formaldehyde treatment, etc.

The binding molecules may also specifically bind to one or morefragments of enterococci (and other Gram-positive and/or Gram-negativebacteria) such as inter alia a preparation of one or more proteinsand/or peptides or polypeptides derived from enterococci or one or morerecombinantly produced enterococcal proteins and/or polypeptides. Formethods of treatment and/or prevention of enterococcal infections thebinding molecules may be capable of specifically binding to surfaceaccessible proteins of enterococci. For diagnostic purposes, the bindingmolecules may also be capable of specifically binding to proteins notpresent on the surface of enterococci. The nucleotide and/or amino acidsequence of proteins of various Enterococcus species and strains can befound in the GenBank-database, EMBL-database and/or other databases.

Alternatively, binding molecules hereof may also be capable ofspecifically binding to other enterococcal molecules including, but notlimited to, surface factors that inhibit phagocytic engulfment; factorsthat enhance their survival in phagocytes; invasions that lyseeukaryotic cell membranes; exotoxins that damage host tissues orotherwise provoke symptoms of disease; polysaccharides; other cell wallcomponents such as teichoic acid, lipoteichoic acid, ribitol,peptidoglycan, pentaglycine oligopeptide, N-acetylglucosamine,N-acetylmuramic acid, N-acetylgalactosaminuronic acid,N-acetylfucosamine, N-acetylglucosaminuronic acid,N-acetylmannosaminuronic acid, O-acetyl, glucosamine, muramic acid,galactosaminuronic acid, fucosamine, glucosaminuronic acid,mannosaminuronic acid rhamnose, hexosamine, hexose, kojibiose, glycerolphosphate, ribitol phosphate and linkage units between any of thesecomponents.

In another embodiment, the binding molecules are able to specificallybind to a fragment of the above-mentioned proteins and/or othermolecules, wherein the fragment at least comprises an antigenicdeterminant recognized by the binding molecules hereof. An “antigenicdeterminant,” as used herein, is a moiety that is capable of binding toa binding molecule hereof with sufficiently high affinity to form adetectable antigen-binding molecule complex.

The binding molecules can be intact immunoglobulin molecules such aspolyclonal or monoclonal antibodies or the binding molecules can beantigen-binding fragments including, but not limited to, Fab, F(ab′),F(ab′)₂, Fv, dAb, Fd, complementarity-determining region (CDR)fragments, single-chain antibodies (scFv), bivalent single-chainantibodies, single-chain phage antibodies, diabodies, triabodies,tetrabodies, and peptides or polypeptides that contain at least afragment of an immunoglobulin that is sufficient to confer specificantigen binding to enterococci or a fragment thereof. In a preferredembodiment the binding molecules hereof are human monoclonal antibodies.

The binding molecules can be used in non-isolated or isolated form.Furthermore, the binding molecules hereof can be used alone or in amixture comprising at least one binding molecule (or variant or fragmentthereof) hereof. In other words, the binding molecules can be used incombination, e.g., as a pharmaceutical composition comprising two ormore binding molecules hereof, variants or fragments thereof. Forexample, binding molecules having different, but complementaryactivities can be combined in a single therapy to achieve a desiredprophylactic, therapeutic or diagnostic effect, but alternatively,binding molecules having identical activities can also be combined in asingle therapy to achieve a desired prophylactic, therapeutic ordiagnostic effect. Optionally, the mixture further comprises at leastone other therapeutic agent. Preferably, the therapeutic agent such as,e.g., an antibiotic is useful in the prophylaxis and/or treatment of anenterococcal infection.

Typically, the binding molecules hereof can bind to their bindingpartners, i.e., enterococci or fragments thereof, with an affinityconstant (K_(d)-value) that is lower than 0.2*10⁻⁴ M, 1.0*10⁻⁵ M,1.0*10⁻⁶ M, 1.0*10⁻⁷ M, preferably lower than 1.0*10⁻⁸ M, morepreferably lower than 1.0*10⁻⁹ M, more preferably lower than 1.0*10⁻¹⁰M, even more preferably lower than 1.0*10⁻¹¹ M, and in particular lowerthan 1.0*10⁻¹² M. The affinity constants can vary for antibody isotypes.For example, affinity binding for an IgM isotype refers to a bindingaffinity of at least about 1.0*10⁻⁷ M. Affinity constants can forinstance be measured using surface plasmon resonance, for example usingthe BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden).

The binding molecules may bind to enterococci or a fragment thereof insoluble form such as for instance in a sample or in suspension or maybind to enterococci or a fragment thereof bound or attached to a carrieror substrate, e.g., microtiter plates, membranes and beads, etc.Carriers or substrates may be made of glass, plastic (e.g.,polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon, etc.The surface of such supports may be solid or porous and of anyconvenient shape. Furthermore, the binding molecules may bind toenterococci in purified/isolated or non-purified/non-isolated form.

Binding molecules exhibit killing activity. Killing activity as meantherein includes, but is not limited to, opsonic activity or any otheractivity increasing/augmenting/enhancing phagocytosis and/or phagocytickilling of bacteria, e.g., enterococci; intrinsic (killing) activity,e.g., reduce or inhibit bacterial growth or directly kill bacteria;increase the sensitivity of bacteria to antibiotic treatment; or anycombination thereof. Opsonic activity can for instance be measured asdescribed herein. Alternative assays measuring opsonic activity aredescribed in for instance Manual of Molecular and Clinical LaboratoryImmunology, 7th Edition. Assays to measure the other mentionedactivities are also known.

In a preferred embodiment, the binding molecules comprise at least aCDR3 region, preferably a heavy chain CDR3 region, comprising the aminoacid sequence selected from the group consisting of SEQ ID NO:3, SEQ IDNO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ IDNO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:196, SEQ IDNO:202, SEQ ID NO:220, SEQ ID NO:226, SEQ ID NO:232, SEQ ID NO:238, SEQID NO:244, SEQ ID NO:250, SEQ ID NO:256, SEQ ID NO:262, SEQ ID NO:268,SEQ ID NO:274, SEQ ID NO:280, SEQ ID NO:286, SEQ ID NO:292, SEQ IDNO:298, SEQ ID NO:304, SEQ ID NO:310, SEQ ID NO:316, SEQ ID NO:322, SEQID NO:328, SEQ ID NO:334, SEQ ID NO:340, and SEQ ID NO:346 of theSequence Listing incorporated herein. The CDR regions of the bindingmolecules hereof are shown in Table 11. CDR regions are according toKabat et al. (1991) as described in Sequences of Proteins ofImmunological Interest. In an embodiment binding molecules may comprisetwo, three, four, five or even all six CDR regions of the bindingmolecules.

In yet another embodiment, the binding molecules comprise a heavy chaincomprising the variable heavy chain of the amino acid sequence selectedfrom the group consisting of SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86,SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:211, SEQ ID NO:213, SEQ IDNO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403, SEQID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID NO:413,SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ IDNO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431, SEQID NO:433, SEQ ID NO:435, and SEQ ID NO:437. In a further embodiment,the binding molecules comprise a light chain comprising the variablelight chain of the amino acid sequence selected from the groupconsisting of SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ IDNO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQID NO:118, SEQ ID NO:120, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:439,SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ IDNO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467,SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ IDNO:477, SEQ ID NO:479, and SEQ ID NO:481. Table 12 specifies the heavyand light chain variable regions of the binding molecule.

Another aspect hereof includes functional variants of the bindingmolecules as defined herein. Such molecules are considered to befunctional variants of a binding molecule hereof, if the variants arecapable of competing for specifically binding to enterococci (or otherGram-positive and/or Gram-negative bacteria) or a fragment thereof withthe parental human binding molecules. In other words, the functionalvariants are still capable of binding to enterococci or a fragmentthereof. Preferably, the functional variants are capable of competingfor specifically binding to at least two (or more) differentEnterococcus species or fragments thereof that are specifically bound bythe parental human binding molecules. Furthermore, molecules areconsidered to be functional variants of a binding molecule hereof, ifthey have killing activity against enterococci, preferably against theat least two (or more) Enterococcus species against which the parentalbinding molecule exhibits killing activity.

In another embodiment, the functional variants of a binding moleculealso have killing activity against other Gram-positive and/orGram-negative bacteria. Functional variants include, but are not limitedto, derivatives that are substantially similar in primary structuralsequence, but which contain, e.g., in vitro or in vivo modifications,chemical and/or biochemical, that are not found in the parental bindingmolecule. Such modifications include inter alia acetylation, acylation,covalent attachment of a nucleotide or nucleotide derivative, covalentattachment of a lipid or lipid derivative, cross-linking, disulfide bondformation, glycosylation, hydroxylation, methylation, oxidation,pegylation, proteolytic processing, phosphorylation, and the like.

Alternatively, functional variants can be binding molecules as definedherein comprising an amino acid sequence containing substitutions,insertions, deletions or combinations thereof of one or more amino acidscompared to the amino acid sequences of the parental binding molecules.Furthermore, functional variants can comprise truncations of the aminoacid sequence at either or both the amino or carboxyl termini.Functional variants may have the same or different, either higher orlower, binding affinities compared to the parental binding molecule butare still capable of binding to enterococci or a fragment thereof. Forinstance, functional variants may have increased or decreased bindingaffinities for enterococci or a fragment thereof compared to theparental binding molecules. Preferably, the amino acid sequences of thevariable regions, including, but not limited to, framework regions,hypervariable regions, in particular the CDR3 regions, are modified.Generally, the light chain and the heavy chain variable regions comprisethree hypervariable regions, comprising three CDRs, and more conservedregions, the so-called framework regions (FRs). The hypervariableregions comprise amino acid residues from CDRs and amino acid residuesfrom hypervariable loops. Functional variants intended to fall withinthe scope hereof have at least about 50% to about 99%, preferably atleast about 60% to about 99%, more preferably at least about 70% toabout 99%, even more preferably at least about 80% to about 99%, mostpreferably at least about 90% to about 99%, in particular at least about95% to about 99%, and in particular at least about 97% to about 99%amino acid sequence homology with the parental human binding moleculesas defined herein. Computer algorithms such as inter alia Gap or Bestfitknown to a person skilled in the art can be used to optimally alignamino acid sequences to be compared and to define similar or identicalamino acid residues. Functional variants can be obtained by altering theparental binding molecules or parts thereof by general molecular biologymethods known in the art including, but not limited to, error-prone PCR,oligonucleotide-directed mutagenesis, site-directed mutagenesis andheavy and/or light chain shuffling. In certain embodiments, thefunctional variants hereof have killing activity against enterococci.The killing activity may either be identical, or be higher or lowercompared to the parental binding molecules. Furthermore, the functionalvariants having killing activity may have a further activity suitable inenterococcal control. Other activities are mentioned above. Henceforth,when the term (human) binding molecule is used, this also encompassesfunctional variants of the (human) binding molecule.

Provided is a panel of useful human monoclonal antibodies that haveopsonic phagocytic killing activity against at least one strain of eachof at least two different Enterococcus species and against at least onestrain of Staphylococcus aureus. The antibodies hereof comprise variableregions of any one of antibodies CR5140 (SEQ ID NOS:395+439), CR5157(SEQ ID NOS:397+441), CR6016 (SEQ ID NOS:88+108), CR6043 (SEQ IDNOS:90+110), CR6050 (SEQ ID NOS:401+445), CR6078 (SEQ ID NOS:96+116),CR6087 (SEQ ID NOS:211+215), CR6089 (SEQ ID NOS:213+217), CR6241 (SEQ IDNOS:98+118), CR6252 (SEQ ID NOS:100+120), CR6388 (SEQ ID NOS:421+465),CR6389 (SEQ ID NOS:423+467), CR6396 (SEQ ID NOS:425+469), CR6402 (SEQ IDNOS:427+471), CR6409 (SEQ ID NOS:429+473), CR6415 (SEQ ID NOS:431+475),CR6421 (SEQ ID NOS:433+477) or CR6429 (SEQ ID NOS:435+479) as disclosedherein, and antibodies comprising variable regions with sequences thatare at least 80%, preferably at least 90%, more preferably at least 95%,identical thereto. Preferably the sequences of the complete antibodiesare at least 80%, more preferably at least 90%, still more preferably atleast 95% identical to the sequences of these antibodies as disclosedherein. These antibodies were all shown have opsonic phagocytic killingactivity against at least two different Enterococcus species (comprisingE. faecalis and E. faecium). Surprisingly, these antibodies were alsoreactive against S. aureus (strain 502, and for some antibodies (CR6252,CR6415, CR6421) it was further shown that they were also reactiveagainst strain Numan of S. aureus, as well as against S. epidemidisstrainRP62A), and thus have a broad specificity and broad potential fortherapeutic use. These antibodies did not bind to LTA of S. aureus,which is one of the main constituents of the cell wall of S. aureus. Incertain embodiments, the antibodies hereof therefore do not specificallybind to LTA of S. aureus. Also provided are compositions comprising atleast 2, 3, 4, 5, or more, of the human monoclonal antibodies hereof. Ofcourse, higher affinity mutants or mutants with other advantageousproperties can be prepared according to routine methods, based on thesequences of the antibodies as disclosed herein. Such improvedantibodies are included within the scope hereof, when the variableregions of heavy and light chain are at least 80%, preferably at least90%, still more preferably at least 95% identical to the sequences ofthe variable regions of the antibodies disclosed herein.

Also provided are immunoconjugates, i.e., molecules comprising at leastone binding molecule as defined herein and further comprising at leastone tag, such as inter alia a detectable moiety/agent. Also contemplatedherein are mixtures of immunoconjugates or mixtures of at least oneimmunoconjugates hereof and another molecule, such as a therapeuticagent or another binding molecule or immunoconjugate. In a furtherembodiment, the immunoconjugates may comprise more than one tag. Thesetags can be the same or distinct from each other and can bejoined/conjugated non-covalently to the binding molecules. The tag(s)can also be joined/conjugated directly to the human binding moleculesthrough covalent bonding. Alternatively, the tag(s) can bejoined/conjugated to the binding molecules by means of one or morelinking compounds. Techniques for conjugating tags to binding moleculesare well-known to the skilled artisan.

The tags of the immunoconjugates hereof may be therapeutic agents, butthey can also be detectable moieties/agents. Tags suitable in therapyand/or prevention may be toxins or functional parts thereof,antibiotics, enzymes, other binding molecules that enhance phagocytosisor immune stimulation. Immunoconjugates comprising a detectable agentcan be used diagnostically to, for example, assess if a subject has beeninfected with an Enterococcus species or monitor the development orprogression of an enterococcal infection as part of a clinical testingprocedure to, e.g., determine the efficacy of a given treatment regimen.However, they may also be used for other detection and/or analyticaland/or diagnostic purposes. Detectable moieties/agents include, but arenot limited to, enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,positron emitting metals, and non-radioactive paramagnetic metal ions.The tags used to label the binding molecules for detection and/oranalytical and/or diagnostic purposes depend on the specificdetection/analysis/diagnosis techniques and/or methods used such asinter alia immunohistochemical staining of (tissue) samples, flowcytometric detection, scanning laser cytometric detection, fluorescentimmunoassays, enzyme-linked immunosorbent assays (ELISAs),radioimmunoassays (RIAs), bioassays (e.g., phagocytosis assays), Westernblotting applications, etc. Suitable labels for thedetection/analysis/diagnosis techniques and/or methods known in the artare well within the reach of the skilled artisan.

Furthermore, the human binding molecules or immunoconjugates hereof canalso be attached to solid supports, which are particularly useful for invitro immunoassays or purification of enterococci or a fragment thereof.Such solid supports might be porous or nonporous, planar, or non-planar.The binding molecules hereof can be fused to marker sequences, such as apeptide to facilitate purification. Examples include, but are notlimited to, the hexa-histidine tag, the hemagglutinin (HA) tag, the myctag or the flag tag. Alternatively, an antibody can be conjugated to asecond antibody to form an antibody heteroconjugate. In another aspect,the binding molecules hereof may be conjugated/attached to one or moreantigens. Preferably, these antigens are antigens which are recognizedby the immune system of a subject to which the binding molecule-antigenconjugate is administered. The antigens may be identical, but may alsodiffer from each other. Conjugation methods for attaching the antigensand binding molecules are well-known in the art and include, but are notlimited to, the use of cross-linking agents. The binding moleculeshereof will bind to enterococci and the antigens attached to the bindingmolecules will initiate a powerful T-cell attack on the conjugate, whichwill eventually lead to the destruction of the enterococci.

Next to producing immunoconjugates chemically by conjugating, directlyor indirectly, via for instance a linker, the immunoconjugates can beproduced as fusion proteins comprising the binding molecules hereof anda suitable tag. Fusion proteins can be produced by methods known in theart such as, e.g., recombinantly by constructing nucleic acid moleculescomprising nucleotide sequences encoding the binding molecules in framewith nucleotide sequences encoding the suitable tag(s) and thenexpressing the nucleic acid molecules.

Also provided is a nucleic acid molecule encoding at least a bindingmolecule, functional variant or immunoconjugate hereof. Such nucleicacid molecules can be used as intermediates for cloning purposes, e.g.,in the process of affinity maturation as described herein. In apreferred embodiment, the nucleic acid molecules are isolated orpurified.

The skilled person will appreciate that functional variants of thesenucleic acid molecules are also intended to be a part hereof. Functionalvariants are nucleic acid sequences that can be directly translated,using the standard genetic code, to provide an amino acid sequenceidentical to that translated from the parental nucleic acid molecules.

Preferably, the nucleic acid molecules encode binding moleculescomprising a CDR3 region, preferably a heavy chain CDR3 region,comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57, SEQ IDNO:196, SEQ ID NO:202, SEQ ID NO:220, SEQ ID NO:226, SEQ ID NO:232, SEQID NO:238, SEQ ID NO:244, SEQ ID NO:250, SEQ ID NO:256, SEQ ID NO:262,SEQ ID NO:268, SEQ ID NO:274, SEQ ID NO:280, SEQ ID NO:286, SEQ IDNO:292, SEQ ID NO:298, SEQ ID NO:304, SEQ ID NO:310, SEQ ID NO:316, SEQID NO:322, SEQ ID NO:328, SEQ ID NO:334, SEQ ID NO:340, and SEQ IDNO:346. In a further embodiment the nucleic acid molecules encodebinding molecules comprising two, three, four, five or even all six CDRregions of the binding molecules hereof.

In another embodiment, the nucleic acid molecules encode bindingmolecules comprising a heavy chain comprising the variable heavy chainof the amino acid sequence selected from the group consisting of SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:211, SEQ ID NO:213, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409,SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ IDNO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, and SEQ IDNO:437. In another embodiment the nucleic acid molecules encode bindingmolecules comprising a light chain comprising the variable light chainof the amino acid sequence selected from the group consisting of SEQ IDNO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:439, SEQ ID NO:441, SEQ IDNO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461,SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ IDNO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ ID NO:479, andSEQ ID NO:481.

Also provided are vectors, i.e., nucleic acid constructs, comprising oneor more nucleic acid molecules hereof. Vectors can be derived fromplasmids such as inter alia F, R1, RP1, Col, pBR322, TOL, Ti, etc.;cosmids; phages such as lambda, lambdoid, M13, Mu, P1, P22, Qβ, T-even,T-odd, T2, T4, T7, etc.; plant viruses. Vectors can be used for cloningand/or for expression of the binding molecules hereof and might even beused for gene therapy purposes. Vectors comprising one or more nucleicacid molecules hereof operably linked to one or moreexpression-regulating nucleic acid molecules are also included herein.Vector choice is dependent upon the recombinant procedures followed andthe host used. Introduction of vectors in host cells can be effected byinter alia calcium phosphate transfection, virus infection, DEAE-dextranmediated transfection, lipofectamin transfection or electroporation.Vectors may be autonomously replicating or may replicate together withthe chromosome into which they have been integrated. Preferably, thevectors contain one or more selection markers. The choice of the markersmay depend on the host cells of choice, although this is not critical tothe invention as is well-known to persons skilled in the art. Theyinclude, but are not limited to, kanamycin, neomycin, puromycin,hygromycin, ZEOCIN®, thymidine kinase gene from herpes simplex virus(HSV-TK), dihydrofolate reductase gene from mouse (dhfr). Vectorscomprising one or more nucleic acid molecules encoding the human bindingmolecules as described above operably linked to one or more nucleic acidmolecules encoding proteins or peptides that can be used to isolate thehuman binding molecules are also covered by the invention. Theseproteins or peptides include, but are not limited to,glutathione-S-transferase, maltose binding protein, metal-bindingpolyhistidine, green fluorescent protein, luciferase andbeta-galactosidase.

Hosts containing one or more copies of the vectors mentioned above arean additional subject hereof. In certain embodiments, the hosts are hostcells. Host cells include, but are not limited to, cells of mammalian,plant, insect, fungal or bacterial origin. Bacterial cells include, butare not limited to, cells from Gram-positive bacteria or Gram-negativebacteria such as several species of the genera Escherichia, such as E.coli, and Pseudomonas. In the group of fungal cells preferably yeastcells are used. Expression in yeast can be achieved by using yeaststrains such as inter alia Pichia pastoris, Saccharomyces cerevisiae andHansenula polymorphs. Furthermore, insect cells such as cells fromDrosophila and Sf9 can be used as host cells. Besides that, the hostcells can be plant cells such as inter alia cells from crop plants suchas forestry plants, or cells from plants providing food and rawmaterials such as cereal plants, or medicinal plants, or cells fromornamentals, or cells from flower bulb crops. Transformed (transgenic)plants or plant cells are produced by known methods, for example,Agrobacterium-mediated gene transfer, transformation of leaf discs,protoplast transformation by polyethylene glycol-induced DNA transfer,electroporation, sonication, microinjection or bolistic gene transfer.Additionally, a suitable expression system can be a baculovirus system.Expression systems using mammalian cells such as Chinese Hamster Ovary(CHO) cells, COS cells, BHK cells, or Bowes melanoma cells are preferredherein. Mammalian cells provide expressed proteins withposttranslational modifications that are most similar to naturalmolecules of mammalian origin. Since the disclosure deals with moleculesthat may have to be administered to humans, a completely humanexpression system would be particularly preferred. Therefore, even morepreferably, the host cells are human cells. Examples of human cells areinter alia HeLa, 911, AT1080, A549, 293 and HEK293T cells. In preferredembodiments, the human producer cells comprise at least a functionalpart of a nucleic acid sequence encoding an adenovirus E1 region inexpressible format. In even more preferred embodiments, the host cellsare derived from a human retina and immortalized with nucleic acidscomprising adenoviral E1 sequences, such as 911 cells or the cell linedeposited at the European Collection of Cell Cultures (ECACC), CAMR,Salisbury, Wiltshire SP4 OJG, Great Britain on 29 Feb. 1996 under number96022940 and marketed under the trademark PER.C6® (PER.C6® is aregistered trademark of Crucell Holland B.V.). For the purposes of thisapplication “PER.C6®” refers to cells deposited under number 96022940 orancestors, passages up-stream or downstream as well as descendants fromancestors of deposited cells, as well as derivatives of any of theforegoing. Production of recombinant proteins in host cells can beperformed according to methods well-known in the art. The use of thecells marketed under the trademark PER.C6® as a production platform forproteins of interest has been described in WO 00/63403 the disclosure ofwhich is incorporated herein by reference in its entirety.

A method of producing the binding molecule is an additional part hereof.The method comprises the steps of a) culturing a host under conditionsconducive to the expression of the binding molecule, and b) optionally,recovering the expressed binding molecule. The expressed bindingmolecules or immunoconjugates can be recovered from the cell freeextract, but preferably they are recovered from the culture medium. Thismethod of producing can also be used to make functional variants of thebinding molecules and/or immunoconjugates hereof. Methods to recoverproteins, such as binding molecules, from cell free extracts or culturemedium are well-known to the person skilled in the art. Bindingmolecules, functional variants and/or immunoconjugates as obtainable bythe above-described method are also a part hereof.

Alternatively, next to the expression in hosts, such as host cells, thebinding molecules and immunoconjugates hereof can be producedsynthetically by conventional peptide synthesizers or in cell-freetranslation systems using RNA nucleic acid derived from DNA moleculeshereof. Binding molecules and immunoconjugates as obtainable by theabove described synthetic production methods or cell-free translationsystems are also a part hereof.

In yet another embodiment, binding molecules hereof can also be producedin transgenic, non-human, mammals such as inter alia rabbits, goats orcows, and secreted into for instance the milk thereof.

In yet another alternative embodiment, the binding molecules hereof,preferably human binding molecules specifically binding to enterococcior a fragment thereof, may be generated by transgenic non-human mammals,such as, for instance, transgenic mice or rabbits, that express humanimmunoglobulin genes. Preferably, the transgenic non-human mammals havea genome comprising a human heavy chain transgene and a human lightchain transgene encoding all or a portion of the human binding moleculesas described above. The transgenic non-human mammals can be immunizedwith a purified or enriched preparation of enterococci or a fragmentthereof. Protocols for immunizing non-human mammals are well establishedin the art. See Using Antibodies: A Laboratory Manual, Edited by: E.Harlow, D. Lane (1998), Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Current Protocols in Immunology, Edited by: J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober(2001), John Wiley & Sons Inc., New York, the disclosures of which areincorporated herein by reference. Immunization protocols often includemultiple immunizations, either with or without adjuvants such asFreund's complete adjuvant and Freund's incomplete adjuvant, but mayalso include naked DNA immunizations. In another embodiment, the humanbinding molecules are produced by B-cells or plasma cells derived fromthe transgenic animals. In yet another embodiment, the human bindingmolecules are produced by hybridomas, which are prepared by fusion ofB-cells obtained from the above-described transgenic non-human mammalsto immortalized cells. B-cells, plasma cells and hybridomas asobtainable from the above-described transgenic non-human mammals andhuman binding molecules as obtainable from the above-describedtransgenic non-human mammals, B-cells, plasma cells and hybridomas arealso a part hereof.

In a further aspect, provided are methods of identifying a bindingmolecule, such as a human binding molecule, e.g., a human monoclonalantibody or fragment thereof, specifically binding to at least twodifferent bacterial organisms or nucleic acid molecules encoding suchbinding molecules and comprises the steps of: (a) contacting acollection of binding molecules on the surface of replicable geneticpackages with a first bacterial organism under conditions conducive tobinding, (b) selecting at least once for a replicable genetic packagebinding to the first bacterial organism, (c) optionally, separating thereplicable genetic package binding to the first bacterial organism fromreplicable genetic packages that do not bind to the first bacterialorganism, contacting the separated replicable genetic packages with asecond bacterial organism under conditions conducive to binding andselecting at least once for a replicable genetic package binding to thesecond bacterial organism, and (d) separating and recovering thereplicable genetic package binding to the first and/or second bacterialorganism from replicable genetic packages that do not bind to the firstand/or second bacterial organism. Of course, the above methods extendedwith selections on third and further bacterial organisms are also parthereof. Another part hereof is a method of identifying a bindingmolecule, such as a human binding molecule, e.g., a human monoclonalantibody or fragment thereof, specifically binding to an enterococcalspecies or nucleic acid molecules encoding such a binding molecule. Sucha method comprises the same steps as the method mentioned above. Areplicable genetic package as used herein can be prokaryotic oreukaryotic and includes cells, spores, yeasts, bacteria, viruses, phageor bacteriophage, ribosomes and polysomes. A preferred replicablegenetic package is a phage. The binding molecules, such as for instancesingle chain Fvs, are displayed on the replicable genetic package, i.e.,they are attached to a group or molecule located at an exterior surfaceof the replicable genetic package. The replicable genetic package is ascreenable unit comprising a binding molecule to be screened linked to anucleic acid molecule encoding the binding molecule. The nucleic acidmolecule should be replicable either in vivo (e.g., as a vector) or invitro (e.g., by PCR, transcription and translation). In vivo replicationcan be autonomous (as for a cell), with the assistance of host factors(as for a virus) or with the assistance of both host and helper virus(as for a phagemid). Replicable genetic packages displaying a collectionof binding molecules is formed by introducing nucleic acid moleculesencoding exogenous binding molecules to be displayed into the genomes ofthe replicable genetic packages to form fusion proteins with endogenousproteins that are normally expressed from the outer surface of thereplicable genetic packages. Expression of the fusion proteins,transport to the outer surface and assembly results in display ofexogenous binding molecules from the outer surface of the replicablegenetic packages.

The selection step(s) in the methods can be performed with bacterialorganisms that are live and still infective or inactivated. Inactivationof bacterial organism may be performed by bacterial inactivation methodswell-known to the skilled artisan such as inter alia treatment with lowpH, i.e., pH 4 for 6 hours to 21 days; treatment with organicsolvent/detergent, i.e., addition of organic solvents and detergents(Triton® X-100 or Tween®-80) to the bacterium; UV/light irradiation;gamma-irradiation; and treatment with relevant antibiotics. Methods totest, if a bacterial organism is still alive, infective and/or viable orpartly or completely inactivated are well-known to the person skilled inthe art. The bacterial organisms used in the above method may benon-isolated, e.g., present in serum and/or blood of an infectedindividual. The bacterial organisms used may also be isolated asdiscrete colonies after overnight culture at 37° C. on a suitable mediumsuch as sheep blood agar.

In certain embodiments, the first and/or second bacterial organisms arein suspension when contacted with the replicable genetic packages.Alternatively, they may also be coupled to a carrier when contact takesplace. In another embodiment the first and second bacterial organismsare from a different bacterial family, e.g., the first is from aGram-negative bacterium and the second is from a Gram-positivebacterium. This way, binding molecules capable of specifically bindingto Gram-positive and Gram-negative bacteria can be found. Preferably,the first and second bacterial organisms are both Gram-positivebacteria. The first and second bacterial organism can both beenterococci. In one embodiment the first and second bacterial organismare different strains from the same bacterial species, e.g., anEnterococcus species such as E. faecalis or E. faecium. This way,species-specific binding molecules can be found that are capable ofspecifically binding to different strains within one species. In anotherembodiment, the first and second bacterial organisms are each a memberof a different Enterococcus species, e.g., the first and secondEnterococcus species are selected from the group consisting of E.faecalis and E. faecium. This way, binding molecules capable ofspecifically binding to different species within one bacterial genus canbe found.

Alternatively, the selection step may be performed in the presence of afragment of the bacterial organisms such as, e.g., cell membranepreparations, cell membrane preparations that have been enzymaticallytreated to remove proteins (e.g., with protease K), cell membranepreparations that have been enzymatically treated to remove carbohydratemoieties (e.g., with periodate), recombinant proteins orpolysaccharides. In yet another embodiment, the selection step may beperformed in the presence of one or more proteins or peptides orpolypeptides derived from the bacterial organisms, fusion proteinscomprising these proteins or peptides or polypeptides, and the like.Extracellularly exposed parts of these proteins can also be used asselection material. The live or inactivated bacterial organisms orfragments thereof may be immobilized to a suitable material before use.Alternatively, live or inactivated bacteria in suspension are used. Inan embodiment the selection can be performed on different materialsderived from bacterial organisms. For instance, the first selectionround can be performed on live or inactivated bacterial organisms insuspension, while the second and third selection round can be performedon recombinant bacterial proteins and polysaccharides, respectively. Ofcourse, other combinations are also contemplated herein. Differentbacterial materials can also be used during one selection/panning step.In a further aspect provided are methods wherein the bacterial organismsused in the selection step(s) are derived from the same or differentgrowth phases of the bacteria, e.g., the lag phase, log phase,stationary phase or death phase. This way, e.g., phase-specificanti-bacterial binding molecules may be found. For instance, the firstbacterial organism may be an E. faecalis in stationary phase, while thesecond bacterial organism is an E. faecalis in log phase or the firstbacterial organism may be an E. faecalis in lag phase, while the secondbacterial organism is an E. faecium in lag phase. Further combinationsare well within the reach of the skilled artisan.

Further provided is a method of obtaining a binding moleculespecifically binding to at least two different bacterial organisms or anucleic acid molecule encoding such a binding molecule, wherein themethod comprises the steps of a) performing the above described methodof identifying binding molecules, and b) isolating from the recoveredreplicable genetic package the binding molecule and/or the nucleic acidmolecule encoding the binding molecule. The collection of bindingmolecules on the surface of replicable genetic packages can be acollection of scFvs or Fabs. Once a new scFv or Fab has been establishedor identified with the above-mentioned method of identifying bindingmolecules or nucleic acid molecules encoding the binding molecules, theDNA encoding the scFv or Fab can be isolated from the bacteria or phagesand combined with standard molecular biological techniques to makeconstructs encoding bivalent scFvs or complete human immunoglobulins ofa desired specificity (e.g., IgG, IgA or IgM). These constructs can betransfected into suitable cell lines and complete human monoclonalantibodies can be produced (see Huls et al., 1999; Boel et al., 2000).

The preferred replicable genetic package is a phage. Phage displaymethods for identifying and obtaining (human) binding molecules, e.g.,(human) monoclonal antibodies, are by now well-established methods knownby the person skilled in the art. They are, e.g., described in U.S. Pat.No. 5,696,108; Burton and Barbas, 1994; de Kruif et al., 1995b; andPhage Display: A Laboratory Manual. Edited by: C F Barbas, D R Burton, JK Scott and G J Silverman (2001), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. All these references are herewith incorporatedherein in their entirety. For the construction of phage displaylibraries, collections of human monoclonal antibody heavy and lightchain variable region genes are expressed on the surface ofbacteriophage, preferably filamentous bacteriophage, particles, in forexample single-chain Fv (scFv) or in Fab format (see de Kruif et al.,1995b). Large libraries of antibody fragment-expressing phages typicallycontain more than 1.0*10⁹ antibody specificities and may be assembledfrom the immunoglobulin V-regions expressed in the B-lymphocytes ofimmunized- or non-immunized individuals. In a specific embodiment hereofthe phage library of binding molecules, preferably scFv phage library,is prepared from RNA isolated from cells obtained from a subject thathas been vaccinated against a bacterium, recently vaccinated against anunrelated pathogen, recently suffered from a chronic or acute bacterialinfection, e.g., enterococcal infection, or from a healthy individual.RNA can be isolated from inter alia bone marrow or peripheral blood,preferably peripheral blood lymphocytes or on isolated B-cells or evenon subpopulations of B-cells. The subject can be an animal vaccinatedagainst a bacterium or an animal that has or has had a bacterialinfection. Preferably, the animal is a human subject that has beenvaccinated against a bacterium or has or has had a chronic bacterialinfection or an acute bacterial infection. Preferably, the human subjecthas recently recovered from the bacterial infection.

Alternatively, phage display libraries may be constructed fromimmunoglobulin variable regions that have been partially assembled invitro to introduce additional antibody diversity in the library(semi-synthetic libraries). For example, in vitro assembled variableregions contain stretches of synthetically produced, randomized orpartially randomized DNA in those regions of the molecules that areimportant for antibody specificity, e.g., CDR regions. Phage antibodiesspecific for bacteria such as enterococci can be selected from thelibrary by exposing the bacteria or material thereof to a phage libraryto allow binding of phages expressing antibody fragments specific forthe bacteria or material thereof. Non-bound phages are removed bywashing and bound phages eluted for infection of E. coli bacteria andsubsequent propagation. Multiple rounds of selection and propagation areusually required to sufficiently enrich for phages binding specificallyto the bacteria or material thereof. If desired, before exposing thephage library to the bacteria or material thereof the phage library canfirst be subtracted by exposing the phage library to non-target materialsuch as bacteria of a different family, species and/or strain orbacteria in a different growth phase or material of these bacteria.These subtractor bacteria or material thereof can be bound to a solidphase or can be in suspension. Phages may also be selected for bindingto complex antigens such as complex mixtures of bacterial proteins orpeptides or polypeptides optionally supplemented with bacterialpolysaccharides or other bacterial material. Host cells expressing oneor more proteins or peptides or polypeptides of bacteria such asenterococci may also be used for selection purposes. A phage displaymethod using these host cells can be extended and improved bysubtracting non-relevant binders during screening by addition of anexcess of host cells comprising no target molecules or non-targetmolecules that are similar, but not identical, to the target, andthereby strongly enhance the chance of finding relevant bindingmolecules. Of course, the subtraction may be performed before, during orafter the screening with bacterial organisms or material thereof. Theprocess is referred to as the Mabstract® process (Mabstract® is aregistered trademark of Crucell Holland B.V., see also U.S. Pat. No.6,265,150 which is incorporated herein by reference).

Also provided are methods of obtaining a binding molecule potentiallyhaving killing activity against a bacterial organism, preferably atleast two different bacterial organisms, wherein the method comprisesthe steps of (a) performing the method of obtaining a binding moleculespecifically binding to at least two different bacterial organisms or anucleic acid molecule encoding such a binding molecule as describedabove, and (b) verifying if the binding molecule isolated has killingactivity against the bacterial organism, preferably the at least twodifferent bacterial organisms. Assays for verifying if a bindingmolecule has killing activity such as opsonic activity are well-known inthe art (see for instance Manual of Molecular and Clinical LaboratoryImmunology, 7th Edition). In a further embodiment, the binding moleculeis also tested for any other activity. Other useful activities arementioned above.

Also disclosed is a binding molecule having killing activity against atleast two, preferably at least three or more, different bacterialorganisms, such as, e.g., enterococci, and being obtainable by themethods as described above. A pharmaceutical composition comprising thebinding molecule, the pharmaceutical composition further comprising atleast one pharmaceutically acceptable excipient is also an aspecthereof. Pharmaceutically acceptable excipients are well-known to theskilled person. The pharmaceutical composition herein may furthercomprise at least one other therapeutic agent. Suitable agents are alsowell-known to the skilled artisan.

Also disclosed are compositions comprising at least one binding moleculepreferably a human monoclonal antibody hereof, at least one functionalvariant thereof, at least one immunoconjugate herein or a combinationthereof. In addition to that, the compositions may comprise inter aliastabilizing molecules, such as albumin or polyethylene glycol, or salts.The salts used may be salts that retain the desired biological activityof the binding molecules and do not impart any undesired toxicologicaleffects. If necessary, the human binding molecules hereof may be coatedin or on a material to protect them from the action of acids or othernatural or non-natural conditions that may inactivate the bindingmolecules.

Also disclosed are compositions that comprise at least one nucleic acidmolecule as defined herein. The compositions may comprise aqueoussolutions such as aqueous solutions containing salts (e.g., NaCl orsalts as described above), detergents (e.g., SDS) and/or other suitablecomponents.

Also disclosed are pharmaceutical compositions comprising at least onebinding molecule such as a human monoclonal antibody hereof (orfunctional fragment or variant thereof), at least one immunoconjugatehereof, at least one composition hereof, or combinations thereof. Thepharmaceutical composition hereof further comprises at least onepharmaceutically acceptable excipient.

In certain embodiments, the pharmaceutical compositions may comprise twoor more binding molecules that have killing activity against a bacterialorganism, e.g., an Enterococcus species. In an embodiment, the bindingmolecules exhibit synergistic killing activity, when used incombination. In other words, the compositions comprise at least twobinding molecules having killing activity, characterized in that thebinding molecules act synergistically in killing a bacterial organismsuch as, e.g., an Enterococcus species. As used herein, the term“synergistic” means that the combined effect of the binding moleculeswhen used in combination is greater than their additive effects whenused individually. The synergistically acting binding molecules may bindto different structures on the same or distinct fragments of thebacterial organism. In an embodiment the binding molecules actingsynergistically in killing a bacterial organism may also be capable ofkilling other bacterial organisms synergistically. A way of calculatingsynergy is by means of the combination index. The concept of thecombination index (CI) has been described by Chou and Talalay, 1984. Thetwo or more binding molecules having synergistic activity have distinctmodes of action. For instance a first binding molecule may haveopsonizing activity, while the second binding molecule has anotheractivity increasing/augmenting/enhancing phagocytosis or a first bindingmolecule may have intrinsic (killing) activity, e.g., reduce or inhibitbacterial growth or directly kill bacteria, while the second bindingmolecule increases the sensitivity of bacteria to antibiotic treatment.Other combinations are contemplated herein.

A pharmaceutical composition hereof can further comprise at least oneother therapeutic, prophylactic and/or diagnostic agent. Preferably, thepharmaceutical composition comprises at least one other prophylacticand/or therapeutic agent. Preferably, the further therapeutic and/orprophylactic agents are agents capable of preventing and/or treating abacterial, e.g., enterococcal, infection and/or a condition resultingfrom such an infection. Therapeutic and/or prophylactic agents include,but are not limited to, anti-bacterial agents. Such agents can bebinding molecules, small molecules, organic or inorganic compounds,enzymes, polynucleotide sequences, anti-microbial peptides, etc. Otheragents that are currently used to treat patients infected with bacterialinfections such as enterococcal infections are antibiotics such asvancomycin, teicoplanin, synergistic combinations including ampicillinor vancomycin and an aminoglycoside or sulbactam, penicillins includingextended spectrum penicillins, carbapenems, macrolides, quinolones,tetracyclines, chloramphenicol, daptomycin, linezolid,quinupristin/dalfopristin. These can be used in combination with thebinding molecules hereof. Agents capable of preventing and/or treatingan infection with bacteria and/or a condition resulting from such aninfection that are in the experimental phase might also be used as othertherapeutic and/or prophylactic agents useful herein.

The binding molecules or pharmaceutical compositions hereof may betested in suitable animal model systems prior to use in humans. Suchanimal model systems include, but are not limited to, murine sepsis andperitonitis models, rat sepsis and endocarditis models, and rabbitendocarditis models.

Typically, pharmaceutical compositions are sterile and stable under theconditions of manufacture and storage. The binding molecules,immunoconjugates, nucleic acid molecules or compositions hereof can bein powder form for reconstitution in the appropriate pharmaceuticallyacceptable excipient before or at the time of delivery. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Alternatively, the binding molecules, immunoconjugates, nucleic acidmolecules or compositions hereof can be in solution and the appropriatepharmaceutically acceptable excipient can be added and/or mixed beforeor at the time of delivery to provide a unit dosage injectable form.Preferably, the pharmaceutically acceptable excipient used herein issuitable to high drug concentration, can maintain proper fluidity and,if necessary, can delay absorption.

The choice of the optimal route of administration of the pharmaceuticalcompositions will be influenced by several factors including thephysico-chemical properties of the active molecules within thecompositions, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For instance, if necessary, the binding moleculeshereof can be prepared with carriers that will protect them againstrapid release, such as a controlled release formulation, includingimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can inter alia be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Furthermore, it may be necessaryto coat the binding molecules with, or co-administer the bindingmolecules with, a material or compound that prevents the inactivation ofthe human binding molecules. For example, the binding molecules may beadministered to a subject in an appropriate carrier, for example,liposomes or a diluent.

The routes of administration can be divided into two main categories,oral and parenteral administration. The preferred administration routeis intravenous.

Oral dosage forms can be formulated inter alia as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard capsules, soft gelatin capsules, syrups or elixirs,pills, dragees, liquids, gels, or slurries. These formulations cancontain pharmaceutically excipients including, but not limited to, inertdiluents, granulating and disintegrating agents, binding agents,lubricating agents, preservatives, coloring, flavoring or sweeteningagents, vegetable or mineral oils, wetting agents, and thickeningagents.

The pharmaceutical compositions hereof can also be formulated forparenteral administration. Formulations for parenteral administrationcan be inter alia in the form of aqueous or non-aqueous isotonic sterilenon-toxic injection or infusion solutions or suspensions. The solutionsor suspensions may comprise agents that are non-toxic to recipients atthe dosages and concentrations employed such as 1,3-butanediol, Ringer'ssolution, Hank's solution, isotonic sodium chloride solution, oils,fatty acids, local anesthetic agents, preservatives, buffers, viscosityor solubility increasing agents, water-soluble antioxidants, oil-solubleantioxidants, and metal chelating agents.

In a further aspect, the binding molecules such as human monoclonalantibodies (functional fragments and variants thereof),immunoconjugates, compositions, or pharmaceutical compositions hereofcan be used as a medicament. So, a method of treatment and/or preventionof a bacterial (Gram-positive and/or Gram-negative), e.g., anenterococcal, infection using the binding molecules, immunoconjugates,compositions, or pharmaceutical compositions hereof is another parthereof. The above-mentioned molecules can inter alia be used in thediagnosis, prophylaxis, treatment, or combination thereof, of abacterial infection. Important clinical infections caused by enterococciinclude, but are not limited to, urinary tract infections,intra-abdominal, pelvic and soft tissue infections, bacteremia,bacterial endocarditis, diverticulitis, meningitis, peritonitis,osteomyelitis, septic arthritis, abscesses, wound infections andpneumonia. They are suitable for treatment of yet untreated patientssuffering from a bacterial infection and patients who have been or aretreated for a bacterial infection. They may be used for patients such ashospitalized infants, infants born prematurely, burn victims, elderlypatients, immunocompromised patients such as those receivingchemotherapy, immunosuppressed patients such as those receivingtransplanted organs, immunodeficient patients, patient undergoing aninvasive procedure, and health care workers. Each administration mayprotect against further infection by the bacterial organism for up tothree or four weeks and/or will retard the onset or progress of thesymptoms associated with the infection. The binding molecules hereof mayalso increase the effectiveness of existing antibiotic treatment byincreasing the sensitivity of the bacterium to the antibiotic, maystimulate the immune system to attack the bacterium in ways other thanthrough opsonization. This activation may result in long lastingprotection to the infection bacterium. Furthermore, the bindingmolecules hereof may directly inhibit the growth of the bacterium orinhibit virulence factors required for its survival during theinfection.

The above-mentioned molecules or compositions may be employed inconjunction with other molecules useful in diagnosis, prophylaxis,and/or treatment. They can be used in vitro, ex vivo or in vivo. Forinstance, the binding molecules such as human monoclonal antibodies (orfunctional variants thereof), immunoconjugates, compositions orpharmaceutical compositions hereof can be co-administered with a vaccineagainst the bacterial organism (if available). Alternatively, thevaccine may also be administered before or after administration of themolecules hereof. Instead of a vaccine, anti-bacterial agents can alsobe employed in conjunction with the binding molecules hereof. Suitableanti-bacterial agents are identified herein.

The molecules are typically formulated in the compositions andpharmaceutical compositions hereof in a therapeutically ordiagnostically effective amount. Alternatively, they may be formulatedand administered separately. For instance the other molecules such asthe anti-bacterial agents may be applied systemically, while the bindingmolecules hereof may be applied intrathecally or intraventricularly.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic response). A suitable dosage range may for instancebe 0.1-100 mg/kg body weight, preferably 0.5-15 mg/kg body weight.Furthermore, for example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. The molecules and compositions herein arepreferably sterile. Methods to render these molecules and compositionssterile are well-known in the art. The other molecules useful indiagnosis, prophylaxis and/or treatment can be administered in a similardosage regimen as proposed for the binding molecules hereof. If theother molecules are administered separately, they may be administered toa patient prior to (e.g., 2 minutes, 5 minutes, 10 minutes, 15 minutes,30 minutes, 45 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours,10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24hours, 2 days, 3 days, 4 days, 5 days, 7 days, 2 weeks, 4 weeks or 6weeks before), concomitantly with, or subsequent to (e.g., 2 minutes, 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 2hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5days, 7 days, 2 weeks, 4 weeks or 6 weeks after) the administration ofone or more of the human binding molecules or pharmaceuticalcompositions hereof. The exact dosing regimen is usually sorted outduring clinical trials in human patients.

Human binding molecules and pharmaceutical compositions comprising thehuman binding molecules are particularly useful, and often preferred,when to be administered to human beings as in vivo therapeutic agents,since recipient immune response to the administered antibody will oftenbe substantially less than that occasioned by administration of amonoclonal murine, chimeric or humanized binding molecule.

In another aspect, described is the use of the binding molecules such askilling human monoclonal antibodies (functional fragments and variantsthereof), immunoconjugates, nucleic acid molecules, compositions orpharmaceutical compositions herein in the preparation of a medicamentfor the diagnosis, prophylaxis, treatment, or combination thereof, of abacterial (Gram-positive and/or Gram-negative), e.g., enterococcalinfection.

Next to that, kits comprising at least one binding molecule such as akilling human monoclonal antibody (functional fragments and variantsthereof), at least one immunoconjugate, at least one nucleic acidmolecule, at least one composition, at least one pharmaceuticalcomposition, at least one vector, at least one host hereof or acombination thereof are also a part hereof. Optionally, theabove-described components of the kits hereof are packed in suitablecontainers and labeled for diagnosis, prophylaxis and/or treatment ofthe indicated conditions. The above-mentioned components may be storedin unit or multi-dose containers as an aqueous, preferably sterile,solution or as a lyophilized, preferably sterile, formulation forreconstitution. The containers may be formed from a variety of materialssuch as glass or plastic and may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper pierceable by a hypodermic injection needle). The kit mayfurther comprise more containers comprising a pharmaceuticallyacceptable buffer. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, syringes, culture medium for one or more of thesuitable hosts and, possibly, even at least one other therapeutic,prophylactic or diagnostic agent. Associated with the kits can beinstructions customarily included in commercial packages of therapeutic,prophylactic or diagnostic products, that contain information about forexample the indications, usage, dosage, manufacture, administration,contra-indications and/or warnings concerning the use of suchtherapeutic, prophylactic or diagnostic products.

The binding molecules hereof may also be used to coat medical devices orpolymeric biomaterials.

Further described is a method of detecting a bacterial organism(Gram-positive and/or Gram-negative) in a sample, wherein the methodcomprises the steps of (a) contacting a sample with a diagnosticallyeffective amount of a binding molecule (functional fragments andvariants thereof) or an immunoconjugate herein, and (b) determiningwhether the binding molecule or immunoconjugate specifically binds to amolecule of the sample. Preferably, the method is used to detect anEnterococcus in a sample. The sample may be a biological sampleincluding, but not limited to blood, serum, urine, tissue or otherbiological material from (potentially) infected subjects, or anon-biological sample such as water, drink, etc. The (potentially)infected subjects may be human subjects, but also animals that aresuspected as carriers of such a bacterial organism might be tested forthe presence of the organism using the human binding molecules orimmunoconjugates hereof. The sample may first be manipulated to make itmore suitable for the method of detection. Manipulation means inter aliatreating the sample suspected to contain and/or containing the bacterialorganism in such a way that the organism will disintegrate intoantigenic components such as proteins, peptides or polypeptides or otherantigenic fragments. Preferably, the human binding molecules orimmunoconjugates hereof are contacted with the sample under conditionswhich allow the formation of an immunological complex between the humanbinding molecules and the bacterial organism or antigenic componentsthereof that may be present in the sample. The formation of animmunological complex, if any, indicating the presence of the bacterialorganism in the sample, is then detected and measured by suitable means.Such methods include, inter alia, homogeneous and heterogeneous bindingimmunoassays, such as radio-immunoassays (RIA), ELISA,immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blotanalyses.

Preferred assay techniques, especially for large-scale clinicalscreening of patient sera and blood and blood-derived products are ELISAand Western blot techniques. ELISA tests are particularly preferred. Foruse as reagents in these assays, the binding molecules orimmunoconjugates hereof are conveniently bonded to the inside surface ofmicrotiter wells. The binding molecules or immunoconjugates hereof maybe directly bonded to the microtiter well. However, maximum binding ofthe binding molecules or immunoconjugates hereof to the wells might beaccomplished by pre-treating the wells with polylysine prior to theaddition of the binding molecules or immunoconjugates hereof.Furthermore, the binding molecules or immunoconjugates hereof may becovalently attached by known means to the wells. Generally, the bindingmolecules or immunoconjugates are used between 0.01 to 100 μg/ml forcoating, although higher as well as lower amounts may also be used.Samples are then added to the wells coated with the binding molecules orimmunoconjugates hereof.

Furthermore, the binding molecules hereof can be used to identifyspecific binding structures of a bacterial organism, e.g., anEnterococcus. The binding structures can be epitopes on proteins and/orpolypeptides. They can be linear, but also structural and/orconformational. In one embodiment, the binding structures can beanalyzed by means of PEPSCAN analysis (see inter alia WO 84/03564, WO93/09872, Slootstra et al., 1996). Alternatively, a random peptidelibrary comprising peptides from a protein of a bacterial organism canbe screened for peptides capable of binding to the binding moleculeshereof. The binding structures/peptides/epitopes found can be used asvaccines and for the diagnosis of bacterial infections. In casefragments other than proteins and/or polypeptides are bound by thebinding molecules binding structures can be identified by massspectrometry, high performance liquid chromatography and nuclearmagnetic resonance.

In a further aspect, provided is a method of screening a bindingmolecule (or a functional fragment or variant thereof) for specificbinding to the same epitope of a bacterial organism (Gram-positiveand/or Gram-negative), e.g., Enterococcus, as the epitope bound by ahuman binding molecule hereof, wherein the method comprises the steps of(a) contacting a binding molecule to be screened, a binding moleculehereof and a bacterial organism or fragment thereof, (b) measure if thebinding molecule to be screened is capable of competing for specificallybinding to the bacterial organism or fragment thereof with the bindingmolecule hereof. In a further step it may be determined, if the screenedbinding molecules that are capable of competing for specifically bindingto the bacterial organism or fragment thereof have killing activity,e.g., opsonic activity. A binding molecule that is capable of competingfor specifically binding to the bacterial organism or a fragment thereofwith the binding molecule hereof is another part hereof. In theabove-described screening method, “specifically binding to the sameepitope” also contemplates specific binding to substantially oressentially the same epitope as the epitope bound by the a bindingmolecule hereof. The capacity to block, or compete with, the binding ofthe binding molecules hereof to the bacterial organism typicallyindicates that a binding molecule to be screened binds to an epitope orbinding site on the bacterial organism that structurally overlaps withthe binding site on the bacterial organism that is immunospecificallyrecognized by the binding molecules hereof. Alternatively, this canindicate that a binding molecule to be screened binds to an epitope orbinding site which is sufficiently proximal to the binding siteimmunospecifically recognized by the binding molecules hereof tosterically or otherwise inhibit binding of the binding molecules hereofto the bacterial organism.

In general, competitive inhibition is measured by means of an assay,wherein an antigen composition, i.e., a composition comprising abacterial organism or fragments thereof, is admixed with referencebinding molecules, i.e., the binding molecules hereof, and bindingmolecules to be screened. Usually, the binding molecules to be screenedare present in excess. Protocols based upon ELISAs and Western blottingare suitable for use in such simple competition studies. By usingspecies or isotype secondary antibodies one will be able to detect onlythe bound reference binding molecules, the binding of which will bereduced by the presence of a binding molecule to be screened thatrecognizes substantially the same epitope. In conducting a bindingmolecule competition study between a reference binding molecule and anybinding molecule to be screened (irrespective of species or isotype),one may first label the reference binding molecule with a detectablelabel, such as, e.g., biotin, an enzymatic, a radioactive or other labelto enable subsequent identification. Binding molecules identified bythese competition assays (“competitive binding molecules” or“cross-reactive binding molecules”) include, but are not limited to,antibodies, antibody fragments and other binding agents that bind to anepitope or binding site bound by the reference binding molecule, i.e., abinding molecule hereof, as well as antibodies, antibody fragments andother binding agents that bind to an epitope or binding sitesufficiently proximal to an epitope bound by the reference bindingmolecule for competitive binding between the binding molecules to bescreened and the reference binding molecule to occur. Preferably,competitive binding molecules hereof will, when present in excess,inhibit specific binding of a reference binding molecule to a selectedtarget species by at least 10%, preferably by at least 25%, morepreferably by at least 50%, and most preferably by at least 75%-90% oreven greater. The identification of one or more competitive bindingmolecules that bind to about, substantially, essentially or at the sameepitope as the binding molecules hereof is a straightforward technicalmatter. As the identification of competitive binding molecules isdetermined in comparison to a reference binding molecule, i.e., abinding molecule hereof, it will be understood that actually determiningthe epitope to which the reference binding molecule and the competitivebinding molecule bind is not in any way required in order to identify acompetitive binding molecule that binds to the same or substantially thesame epitope as the reference binding molecule.

EXAMPLES

The following illustrative Examples further describe the application.

Example 1 Construction of scFv Phage Display Libraries Using RNAExtracted from Donors Screened for Opsonic Activity

Samples of blood were taken from donors reporting a recent gram-positivebacterial infection as well as healthy adults between 25-50 years ofage. Peripheral blood leukocytes were isolated by centrifugation and theblood serum was saved and frozen at −80° C. Donor serum was screened forkilling activity using an opsonophagocytic killing assay (Huebner et al.1999) and compared to normal rabbit serum. Sera from donors havingphagocytic activity greater than the normal serum were chosen to use forthe generation of phage display libraries. Total RNA was prepared fromthe peripheral blood leukocytes of these donors using organic phaseseparation and subsequent ethanol precipitation. The obtained RNA wasdissolved in RNAse free water and the concentration was determined by OD260 nm measurement. Thereafter, the RNA was diluted to a concentrationof 100 ng/μl. Next, 1 μg of RNA was converted into cDNA as follows: To10 μl total RNA, 13 μl DEPC-treated ultrapure water and 1 μl randomhexamers (500 ng/μl) were added and the obtained mixture was heated at65° C. for 5 minutes and quickly cooled on wet-ice. Then, 8 μl 5×First-Strand buffer, 2 μl dNTP (10 mM each), 2 μl DTT (0.1 M), 2 μlRNAse-inhibitor (40 U/μl) and 2 μl SUPERSCRIPT™ III MMLV reversetranscriptase (200 U/μl) were added to the mixture, incubated at roomtemperature for 5 minutes and incubated for 1 hour at 50° C. Thereaction was terminated by heat inactivation, i.e., by incubating themixture for 15 minutes at 75° C. The obtained cDNA products were dilutedto a final volume of 200 μl with DEPC-treated ultrapure water. The OD260 nm of a 50 times diluted solution (in 10 mM Tris buffer) of thedilution of the obtained cDNA products was used to determine the cDNAconcentration. For each donor 5 to 10 μl of the diluted cDNA productswere used as template for PCR amplification of the immunoglobulin gammaheavy chain family and kappa or lambda light chain sequences usingspecific oligonucleotide primers (see Tables 1-7). In addition, for onedonor PCR amplification of the immunoglobulin mu heavy chain family andkappa or lambda light chain sequences was carried out. PCR reactionmixtures contained, besides the diluted cDNA products, 25 pmol senseprimer and 25 pmol anti-sense primer in a final volume of 50 μl of 20 mMTris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 250 μM dNTPs and 1.25 unitsTaq polymerase. In a heated-lid thermal cycler having a temperature of96° C., the mixtures obtained were quickly melted for 2 minutes,followed by 30 cycles of: 30 seconds at 96° C., 30 seconds at 55° C. or60° C. and 60 seconds at 72° C. Finally, the samples were incubated 10minutes at 72° C. and refrigerated at 4° C. until further use.

In a first round amplification, each of 18 light chain variable regionsense primers (twelve for the lambda light chain (see Table 1; theHuVL1A-Back, HuVL1B-Back and HuVL1C-Back sense primers were mixed toequimolarity before use, as well as the HuVL9-Back and HuVL10-Back senseprimers) and six for the kappa light chain (see Table 2)) were combinedwith an anti-sense primer recognizing the C-kappa constant region calledHuCK-FOR 5′-ACACTCTCCCCTGTTGAAGCTCTT-3′ (see SEQ ID NO:121) or C-lambdaconstant region HuCL2-FOR 5′-TGAACATTCTGTAGGGGCCACTG-3′ (see SEQ IDNO:122) and HuCL7-FOR 5′-AGAGCATTCTGCAGGGGCCACTG-3′ (see SEQ ID NO:123)(the HuCL2-FOR and HuCL7-FOR anti-sense primers were mixed toequimolarity before use), yielding 15 products of about 650 base pairs.These products were purified on agarose gel and isolated from the gelusing Qiagen gel-extraction columns. 1/10 of each of the isolatedproducts was used in an identical PCR reaction as described above usingeighteen sense primers, whereby each lambda light chain sense primer wascombined with one of the three Jlambda-region specific anti-senseprimers and each kappa light chain sense primer was combined with one ofthe five Jkappa-region specific anti-sense primers (see Table 3; theHuVL1A-Back-SAL, HuVL1B-Back-SAL and HuVL1C-Back-SAL sense primers weremixed to equimolarity before use, as well as the HuVL9-Back-SAL andHuVL10-Back-SAL sense primers). The sense primers used in the secondamplification were the same primers as used in the first amplification,but extended with restriction sites (see Table 3) to enable directedcloning in the phage display vector PDV-006 (see SEQ ID NO:124). Thisresulted in 57 products of approximately 400 base pairs that were pooledas shown in Table 4 to maintain the natural distribution of thedifferent J segments and light chain families within the library and notto over or under represent certain families. The pooled products werepurified using Qiagen PCR purification columns. In the next step, 3 μgof pooled products and 100 μg PDV-006 vector were digested with SalI andNotI and purified from gel. Thereafter, a ligation was performedovernight at 16° C. as follows. To 500 ng PDV-006 vector either 35, 70or 140 ng pooled products were added in a total volume of 50 μl ligationmix containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mMATP, 25 μg/ml BSA and 2.5 μl T4 DNA Ligase (400 U/μl). The ligationmixes were purified by phenol/chloroform extraction, followed by achloroform extraction and ethanol precipitation, methods well-known tothe skilled artisan. The DNA obtained was dissolved in 50 μl 10 mMTris-HCl pH8.5 and per ligation mix 1 or 2 μl was electroporated into 40μl of TG1 competent E. coli bacteria according to the manufacturer'sprotocol (Stratagene). Transformants were grown overnight at 37° C. on2TY agar supplemented with 50 μg/ml ampicillin and 4.5% glucose.Colonies were counted to determine the optimal vector to insert ratio.From the ligation mix with the optimal ratio, multiple 1 or 2 μlaliquots were electroporated as above and transformants were grownovernight at 37° C., typically yielding ˜10⁷ colonies. A library orsublibrary of variable light chain regions was obtained by scraping thetransformants from the agar plates. This library or sublibrary wasdirectly used for plasmid DNA preparation using a QIAGEN™ QIAFilter MAXIprep kit.

Heavy chain immunoglobulin sequences were amplified from the same cDNApreparations in a similar two round PCR procedure and identical reactionparameters as described above for the light chain regions with theproviso that the primers depicted in Tables 5 and 6 were used. The firstamplification was performed using a set of eight sense directed primers(see Table 5; the HuVH1B/7A-Back and HuVH1C-Back sense primers weremixed to equimolarity before use) each combined with an IgG specificconstant region anti-sense primer called HuCIgG 5′-GTC CAC CTT GGT GTTGCT GGG CTT-3′ (SEQ ID NO:125) yielding seven products of about 650 basepairs. For one donor an IgM specific constant region anti-sense primercalled HuCIgM 5′-TGG AAG AGG CAC GTT CTT TTC TTT-3′ (SEQ ID NO:126) wasused instead of primer HuCIgG. The products were purified on agarose geland isolated from the gel using Qiagen gel-extraction columns. 1/10 ofeach of the isolated products was used in an identical PCR reaction asdescribed above using eight sense primers, whereby each heavy chainsense primer was combined with one of the four JH-region specificanti-sense primers (see Table 6; the HuVH1B/7A-Back-Sfi andHuVH1C-Back-Sfi sense primers were mixed to equimolarity before use).The sense primers used in the second round were the same primers as usedin the first amplification, but extended with restriction sites (seeTable 6) to enable directed cloning in the light chain (sub)libraryvector. This resulted in 28 products of approximately 400 base pairsthat were pooled as shown in Table 7 to maintain the naturaldistribution of the different J segments and heavy chain families withinthe library and not to over or under represent certain families. Thepooled products were purified using Qiagen PCR purification columns.Next, 3 μg of purified products was digested with SfiI and XhoI andligated in the light chain (sub)library vector, which was cut with thesame restriction enzymes, using the same ligation procedure and volumesas described above for the light chain (sub)library. Ligation mixpurification and subsequent transformation of the resulting definitivelibrary was also performed as described above for the light chain(sub)library. All bacteria, typically ˜10⁷, were harvested in 2TYculture medium containing 50 μg/ml ampicillin and 4.5% glucose, mixedwith glycerol to 15% (v/v) and frozen in 1.5 ml aliquots at −80° C.Rescue and selection of each library were performed as described below.The various libraries were named GPB-05-M01, GPB-05-G01, GPB-05-G02,GPB-05-G03, GPB-05-G04 and GPB-05-G05. Two other libraries, RAB-03-G01and RAB-04-G01, were constructed using a method similar to the procedureabove, as described previously in international patent application WO2005/118644.

Example 2 Construction of scFv Phage Display Libraries Using RNAExtracted from Memory B Cells

Peripheral blood was collected from normal healthy donors, convalescentdonors or vaccinated donors by venapunction using EDTA anti-coagulationsample tubes. A blood sample (45 ml) was diluted twice with PBS and 30ml aliquots were underlayed with 10 ml Ficoll-Hypaque (Pharmacia) andcentrifuged at 900×g for 20 minutes at room temperature without breaks.The supernatant was removed carefully to just above the white layercontaining the lymphocytic and thrombocytic fraction. Next, this layerwas carefully removed (˜10 ml), transferred to a fresh 50 ml tube andwashed three times with 40 ml PBS and spun at 400×g for 10 minutes atroom temperature to remove thrombocytes. The obtained pellet containinglymphocytes was resuspended in RPMI medium containing 2% FBS and thecell number was determined by cell counting. Approximately 1×10⁸lymphocytes were stained for fluorescent cell sorting using CD24, CD27and surface IgM as markers for the isolation of switched and IgM memoryB cells. A Becton Dickinson Digital Vantage apparatus set in Yield Modewas used for physical memory B cell sorting and isolation. Lymphocyteswere gated as the small compact population from the FSC/SSC window.Memory B cells (CD24+/CD27+) were subsequently separated from naive Bcells (CD24+/CD27−) and memory T cells (CD24−/CD27+). In a next step,IgM memory B cells (IgM+) were separated from switch memory B cells(IgM−) using IgM expression. In this step IgM memory B cells and switchmemory B cells were sorted in separate sample tubes. 1×10⁵ to 1×10⁶cells of each population were collected in DMEM/50% FBS and aftercompletion of the sort they were each centrifuged at 400×g for 10minutes. The sorted IgM memory B cells were then used as startingmaterial for library construction according to the method described inExample 1, using primer HuCIgM in the first round amplification of heavychain immunoglobulin sequences. The various libraries were namedMEM-05-M01, MEM-05-M02, MEM-05-M03, MEM-05-M04, MEM-05-M05, MEM-05-M06,MEM-05-M07, MEM-05-M08, MEM-05-M09 and MEM-05-M10.

Example 3 Selection of Phages Carrying Single Chain Fv FragmentsSpecifically Binding to Enterococci

Antibody fragments were selected using antibody phage display libraries,general phage display technology and MAbstract® technology, essentiallyas described in U.S. Pat. No. 6,265,150 and in WO 98/15833 (both ofwhich are incorporated by reference herein). The antibody phagelibraries used were screened and donor libraries prepared as describedin Example 1 and IgM memory libraries prepared as described in Example2. The methods and helper phages as described in WO 02/103012(incorporated by reference herein) were used herein. For identifyingphage antibodies recognizing enterococci, phage selection experimentswere performed using live bacteria in suspension or bacteria immobilizedin immunotubes. Strains used are described in Table 8. All phageantibodies were isolated from selections wherein in at least one step E.faecalis 12030 in suspension was used. The phage antibodies calledSC05-159 and SC05-166 were originally isolated from selections usingimmobilized E. faecalis 12030, and isolated using E. faecalis 12030 insuspension.

Selections using bacteria in suspension were performed as follows.Bacteria were grown overnight at 37° C. on blood agar plates and scrapedinto PBS containing 2% BSA or 2% ELK at a concentration of 5×10⁹bacteria/ml and incubated for 30 minutes at room temperature. An aliquotof a phage library (approximately 10¹³ cfu, amplified using CT helperphage (see WO 02/103012)) was blocked in blocking buffer (2% ELK or 2%BSA in PBS) for 0.5-2 hours at room temperature. The blocked phagelibrary was added to the blocked bacterial suspension making a totalvolume of 1 ml and incubated for 2 hours at room temperature in anend-over-end rotor (5 rpm). The suspension was centrifuged at 6800×g for3 minutes at room temperature and the supernatant was discarded.Bacteria were washed three to eight times with blocking buffercontaining 0.05% (v/v) Tween-20, then three to eight times with blockingbuffer to remove unbound phages. Bound phages were eluted from theantigen by incubation with 1 ml of 0.1 M triethylamine for 7 minutes atroom temperature in an end-over-end rotor (5 rpm). The suspension wascentrifuged at 1700×g for 3 minutes at room temperature and thesupernatant was then mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 toneutralize the pH. This mixture was used to infect 5 ml of an XL1-BlueE. coli culture that had been grown at 37° C. to an OD 600 nm ofapproximately 0.3. The phages were allowed to infect the XL1-Bluebacteria for 30 minutes at 37° C. Then, the mixture was centrifuged for10 minutes at 3200×g at room temperature and the bacterial pellet wasresuspended in 0.5 ml 2-trypton yeast extract (2TY) medium. The obtainedbacterial suspension was divided over two 2TY agar plates supplementedwith tetracyclin, ampicillin and glucose. After overnight incubation ofthe plates at 37° C., the colonies were scraped from the plates and usedto prepare an enriched phage library, essentially as described by DeKruif et al. (1995a) and WO 02/103012. Briefly, scraped bacteria wereused to inoculate 2TY medium containing ampicillin, tetracycline andglucose and grown at a temperature of 37° C. to an OD 600 nm of ˜0.3. CThelper phages were added and allowed to infect the bacteria after whichthe medium was changed to 2TY containing ampicillin, tetracycline andkanamycin. Incubation was continued overnight at 30° C. The next day,the bacteria were removed from the 2TY medium by centrifugation afterwhich the phages in the medium were precipitated using polyethyleneglycol (PEG) 6000/NaCl. Finally, the phages were dissolved in 2 ml ofPBS with 1% bovine serum albumin (BSA), filter-sterilized and used forthe next round of selection.

Selections using bacteria immobilized in immunotubes were performed asfollows. Bacteria were grown overnight at 37° C. on blood agar platesand scraped into carbonate buffer at a concentration of 5×10⁹bacteria/ml. Two ml was added to a MaxiSorp™ Nunc-Immuno Tube (Nunc) andincubated overnight at 4° C. in an end-over-end rotor (5 rpm). The tubewas emptied and washed three times with PBS. Both the tube and analiquot of a phage library (approximately 10¹³ cfu, amplified using CThelper phage (see WO 02/103012)) were blocked in blocking buffer (2%ELK, 2% BSA or 1% Protifar in PBS) for 0.5-2 hours at room temperature.The tube was emptied, the blocked phage library was added and the tubewas incubated for 2 hours at room temperature in an end-over-end rotor(5 rpm). The tube was washed five to fifteen times with PBS containing0.1% (v/v) Tween-20, then five to fifteen times with PBS to removeunbound phages. Bound phages were eluted from the antigen by incubationwith 1.5 ml of 0.1 M triethylamine or 50 mM Glycine-HCl, pH 2.2 for 10minutes at room temperature in an end-over-end rotor (5 rpm). The elutedphages were mixed with 0.5 ml of 1 M Tris-HCl pH 7.5 to neutralize thepH. Subsequent infection of XL1-Blue E. coli bacteria, growth ofinfected bacteria and preparation of an enriched phage library wasperformed as described above for selections using bacteria insuspension.

Typically, two rounds of selections were performed before isolation ofindividual phage antibodies. Selection could be carried out twice on thesame strain of bacteria or different strains could be used sequentially.After the second round of selection, individual E. coli colonies wereused to prepare monoclonal phage antibodies. Essentially, individualcolonies were grown to log-phase and infected with CT or VCSM13 helperphages after which phage antibody production was allowed to proceedovernight. The produced phage antibodies were PEG/NaCl-precipitated andfilter-sterilized and tested in ELISA for binding to Enterococcusprepared as described supra.

Example 4 Validation of the Enterococcal Specific Single-Chain PhageAntibodies

Selected single-chain phage antibodies that were obtained in the screensdescribed above were validated in ELISA for specific enterococcalbinding activity, i.e., binding to one or more enterococcal strainsprepared as described supra. 2.5×10⁸ bacteria were coated overnight at4° C. to Maxisorp™ ELISA plates in 50 μl 50 mM carbonate buffer, pH 9.6.As negative controls, the complex antigens 2% ELK and 1% BSA both in PBS(pH 7.4) were coated. Wells were washed in PBS containing 0.1% (v/v)Tween-20 and blocked with 300 μl PBS containing 2% ELK for at least 1hour at room temperature. The selected single-chain phage antibodieswere incubated for 15 minutes in an equal volume of PBS containing 2%ELK to obtain blocked phage antibodies. The plates were emptied and theblocked single-chain phage antibodies were added to the wells.Incubation was allowed to proceed for one hour at room temperature, theplates were washed in PBS containing 0.1% (v/v) Tween-20 and bound phageantibodies were detected using an anti-M13 antibody conjugated toperoxidase. Absorbance at 492 nm was measured using a spectrophotometer.As a control, the procedure was performed simultaneously withoutsingle-chain phage antibody and with a negative control single-chainphage antibody directed against West Nile virus envelope protein(SC04-374). As shown in Table 9, the selected phage antibodies calledSC05-140, SC05-157, SC05-159, SC05-166, SC05-179, SC05-187, SC06-016,SC06-043, SC06-049, SC06-050, SC06-071, SC06-077, SC06-078, SC06-079,SC06-086, SC06-087, SC06-089, SC06-092, SC06-191, SC06-195, SC06-198,SC06-241, SC06-242, SC06-246, SC06-252, SC06-388, SC06-389, SC06-396,SC06-402, SC06-409, SC06-415, SC06-421, SC06-429 and SC06-432specifically bound to Enterococcus faecalis strain 12030. With theexception of SC05-140 and SC06-421 none of the selected phage antibodiesdid display any detectable binding to the negative control antigens ELKand BSA.

Example 5 Characterization of the Enterococcal Specific scFvs

From the selected specific single-chain phage antibody (scFv) clonesplasmid DNA was obtained and nucleotide sequences were determinedaccording to standard techniques. The nucleotide sequences of the scFvs(including restriction sites for cloning) called SC05-140, SC05-157,SC05-159, SC05-166, SC05-179, SC05-187, SC06-016, SC06-043, SC06-049,SC05-050, SC06-071, SC06-077, SC06-078, SC06-079, SC06-086, SC06-087,SC06-089, SC06-092, SC06-191, SC06-195, SC06-198, SC06-241, SC06-242,SC06-246, SC06-252, SC06-388, SC06-389, SC06-396, SC06-402, SC06-409,SC06-415, SC06-421, SC06-429, and SC06-432 are shown in SEQ ID NO:350,SEQ ID NO:352, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:354, SEQ ID NO:65,SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:356, SEQ ID NO:73,SEQ ID NO:358, SEQ ID NO:75, SEQ ID NO:360, SEQ ID NO:362, SEQ IDNO:206, SEQ ID NO:208, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQID NO:370, SEQ ID NO:77, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:79, SEQID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384,SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390 and SEQ ID NO:392,respectively. The amino acid sequences of the scFvs called SC05-140,SC05-157, SC05-159, SC05-166, SC05-179, SC05-187, SC06-016, SC06-043,SC06-049, SC05-050, SC06-071, SC06-077, SC06-078, SC06-079, SC06-086,SC06-087, SC06-089, SC06-092, SC06-191, SC06-195, SC06-198, SC06-241,SC06-242, SC06-246, SC06-252, SC06-388, SC06-389, SC06-396, SC06-402,SC06-409, SC06-415, SC06-421, SC06-429, and SC06-432 are shown in SEQ IDNO:351, SEQ ID NO:353, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:355, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:357, SEQ IDNO:74, SEQ ID NO:359, SEQ ID NO:76, SEQ ID NO:361, SEQ ID NO:363, SEQ IDNO:207, SEQ ID NO:209, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQID NO:371, SEQ ID NO:78, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:80, SEQID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:385,SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391 and SEQ ID NO:393,respectively. The VH and VL gene identity (see Tomlinson I M, Williams SC, Ignatovitch O, Corbett S J, Winter G. V-BASE Sequence Directory.Cambridge United Kingdom: MRC Centre for Protein Engineering (1997)) andCDR sequences of the scFvs specifically binding enterococci are depictedin Tables 10 and 11, respectively.

Example 6 Construction of Fully Human Immunoglobulin Molecules (HumanMonoclonal Anti-Enterococcal Antibodies) from the SelectedAnti-Enterococcal Single Chain Fvs

Heavy and light chain variable regions of the scFv called SC05-140,SC05-157, SC05-159, SC05-166, SC05-179, SC05-187, SC06-016, SC06-043,SC06-049, SC05-050, SC06-071, SC06-077, SC06-078, SC06-079, SC06-086,SC06-087, SC06-089, SC06-092, SC06-191, SC06-195, SC06-198, SC06-241,SC06-242, SC06-246, SC06-252, SC06-388, SC06-389, SC06-396, SC06-402,SC06-409, SC06-415, SC06-421, SC06-429, and SC06-432 were cloneddirectly by restriction digest for expression in the IgG expressionvectors pIg-C911-HCgamma1 (see SEQ ID NO:127), pIg-C909-Ckappa (see SEQID NO:128) and pIg-C910-Clambda (see SEQ ID NO:129). The heavy chainvariable regions of the scFvs called SC05-140, SC05-157, SC05-159,SC05-166, SC05-179, SC05-187, SC06-016, SC06-043, SC06-049, SC05-050,SC06-071, SC06-077, SC06-078, SC06-079, SC06-086, SC06-087, SC06-089,SC06-092, SC06-191, SC06-195, SC06-198, SC06-241, SC06-242, SC06-246,SC06-252, SC06-388, SC06-389, SC06-396, SC06-402, SC06-409, SC06-415,SC06-421, SC06-429, and SC06-432 were cloned into the vectorpIg-C911-HCgamma1 by restriction digest using the enzymes SfiI and XhoI.The light chain variable region of the scFv called SC06-016, SC06-050,SC06-077, SC06-086, SC06-191, SC06-241, SC06-396, and SC06-429 werecloned into the vector pIg-C909-Ckappa by restriction digest using theenzymes SalI, XhoI and NolI. The light chain variable region of the scFvcalled SC05-140, SC05-157, SC05-159, SC05-166, SC05-179, SC05-187,SC06-043, SC06-049, SC06-071, SC06-078, SC06-079, SC06-087, SC06-089,SC06-092, SC06-195, SC06-198, SC06-242, SC06-246, SC06-252, SC06-388,SC06-389, SC06-402, SC06-409, SC06-415, SC06-421, and SC06-432 werecloned into the vector pIg-C910-Clambda by restriction digest using theenzymes SalI, XhoI and NolI. Thereafter the nucleotide sequences wereverified according to standard techniques.

The resulting expression pgG105-140C911, pgG105-157C911, pgG105-159C911,pgG105-166C911, pgG105-179C911, pgG105-187C911, pgG106-016C911,pgG106-043C911, pgG106-049C911, pgG106-050C911, pgG106-071C911,pgG106-077C911, pgG106-078C911, pgG106-079C911, pgG106-086C911,pgG106-087C911, pgG106-089C911, pgG106-092C911, pgG106-191C911,pgG106-195C911, pgG106-198C911, pgG106-0241C911, pgG106-242C911,pgG106-246C911, pgG106-252C911, pgG106-388C911, pgG106-389C911,pgG106-396C911, pgG106-402C911, pgG106-409C911, pgG106-415C911,pgG106-421C911, pgG106-429C911, and pgG106-432C911 encodinganti-enterococci human IgG1 heavy chains and pgG105-140C910,pgG105-157C910, pgG105-159C910, pgG105-166C910, pgG105-179C910,pgG105-187C910, pgG106-016C909, pgG106-043C910, pgG106-049C910,pgG106-050C909, pgG106-071C910, pgG106-077C909, pgG106-078C910,pgG106-079C910, pgG106-086C909, pgG106-087C910, pgG106-089C910,pgG106-092C910, pgG106-191C909, pgG106-195C910, pgG106-198C910,pgG106-0241C909, pgG106-242C910, pgG106-246C910, pgG106-252C910,pgG106-388C910, pgG106-389C910, pgG106-396C909, pgG106-402C910,pgG106-409C910, pgG106-415C910, pgG106-421C910, pgG106-429C909, andpgG106-432C910 encoding the anti-enterococci human Ig light chains weretransiently expressed in combination in 293T cells and supernatantscontaining human IgG1 antibodies were obtained. The nucleotide sequencesof the heavy chains of the antibodies called CR5140, CR5157, CR5159,CR5166, CR5179, CR5187, CR6016, CR6043, CR6049, CR6050, CR6071, CR6077,CR6078, CR6079, CR6086, CR6087, CR6089, CR6092, CR6191, CR6195, CR6198,CR6241, CR6242, CR6246, CR6252, CR6388, CR6389, CR6396, CR6402, CR6409,CR6415, CR6421, CR6429, and CR6432 are shown in SEQ ID NO:394, SEQ IDNO:396, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:398, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:400, SEQ ID NO:93, SEQ IDNO:402, SEQ ID NO:95, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:210, SEQID NO:212, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414,SEQ ID NO:97, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:99, SEQ ID NO:420,SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ IDNO:430, SEQ ID NO:432, SEQ ID NO:434, and SEQ ID NO:436, respectively.The amino acid sequences of the heavy chains of the antibodies calledCR5140, CR5157, CR5159, CR5166, CR5179, CR5187, CR6016, CR6043, CR6049,CR6050, CR6071, CR6077, CR6078, CR6079, CR6086, CR6087, CR6089, CR6092,CR6191, CR6195, CR6198, CR6241, CR6242, CR6246, CR6252, CR6388, CR6389,CR6396, CR6402, CR6409, CR6415, CR6421, CR6429, and CR6432, are shown inSEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:399,SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:401,SEQ ID NO:94, SEQ ID NO:403, SEQ ID NO:96, SEQ ID NO:405, SEQ ID NO:407,SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:409, SEQ ID NO:411, SEQ IDNO:413, SEQ ID NO:415, SEQ ID NO:98, SEQ ID NO:417, SEQ ID NO:419, SEQID NO:100, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427,SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, and SEQ IDNO:437, respectively.

The nucleotide sequences of the light chain of antibodies CR5140,CR5157, CR5159, CR5166, CR5179, CR5187, CR6016, CR6043, CR6049, CR6050,CR6071, CR6077, CR6078, CR6079, CR6086, CR6087, CR6089, CR6092, CR6191,CR6195, CR6198, CR6241, CR6242, CR6246, CR6252, CR6388, CR6389, CR6396,CR6402, CR6409, CR6415, CR6421, CR6429, and CR6432 are shown in SEQ IDNO:438, SEQ ID NO:440, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:442, SEQID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:444,SEQ ID NO:113, SEQ ID NO:446, SEQ ID NO:115, SEQ ID NO:448, SEQ IDNO:450, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:452, SEQ ID NO:454, SEQID NO:456, SEQ ID NO:458, SEQ ID NO:117, SEQ ID NO:460, SEQ ID NO:462,SEQ ID NO:119, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ IDNO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, andSEQ ID NO:480, respectively. The amino acid sequences of the light chainof antibodies CR5140, CR5157, CR5159, CR5166, CR5179, CR5187, CR6016,CR6043, CR6049, CR6050, CR6071, CR6077, CR6078, CR6079, CR6086, CR6087,CR6089, CR6092, CR6191, CR6195, CR6198, CR6241, CR6242, CR6246, CR6252,CR6388, CR6389, CR6396, CR6402, CR6409, CR6415, CR6421, CR6429, andCR6432 are shown in SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:443, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:445, SEQ ID NO:114, SEQ ID NO:447, SEQ ID NO:116,SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:118, SEQID NO:461, SEQ ID NO:463, SEQ ID NO:120, SEQ ID NO:465, SEQ ID NO:467,SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ IDNO:477, SEQ ID NO:479, and SEQ ID NO:481, respectively. One candetermine the variable regions of the heavy and light chains of theabove antibodies by following Kabat et al. (1991) as described inSequences of Proteins of Immunological Interest. Table 12 provides thevariable regions of the antibodies.

The human anti-enterococcal IgG1 antibodies were validated for theirability to bind enterococci by ELISA essentially as described for scFvsabove; IgG1 were assayed at a concentration of 5 μg/ml except for thefollowing IgG1s: CR6191 was assayed at 1.6 μg/ml, CR6195 at 3.1 μg/ml,CR6198 at 4.1 μg/ml, CR6241 at 2.7 μg/ml, CR6246 at 2.6 μg/ml and CR6252at 3.0 μg/ml. The negative control was an anti-West Nile virus antibody(CR4374). In addition, the human anti-enterococcal IgG1 antibodies weretested for their ability to bind to different clinical isolates ofEnterococcus faecalis and Enterococcus faecium (see Table 13). Anantibody was considered to bind to an isolate, when the value within anindividual experiment was at least three-fold compared to the value ofthe negative control within that individual experiment. The value of thenegative control in Table 13 is an average of 6 experiments. Allantibodies except CR5157, CR5179, CR6016, CR6043, CR6050, CR6246,CR6388, CR6409 and the negative control antibody specifically bound toE. faecalis strain 12030 and all IgG1s with the exception of CR5157,CR6016, CR6043, CR6050, CR6241, CR6242, CR6246, CR6388 and CR6409 boundto more than one clinical isolate. Antibodies CR5187, CR6049, CR6396,CR6402 and CR6421 bound to all E. faecalis strains tested and the two E.faecium strains tested. Alternatively, batches of greater than 1 mg ofeach antibody were produced and purified using standard procedures.

Example 7 In Vitro Opsonic Phagocytic Activity of Enterococcal SpecificIgGs Measured by Opsonophagocytic Killing Assay

An opsonophagocytic assay was conducted to quantify the killing activityof anti-enterococci human IgG1 against the enterococcal clinical isolate12030. Freshly drawn human blood (10 to 30 ml) was mixed with an equalvolume of dextran-heparin buffer (4.5 g of dextran, Sigma Chemical, St.Louis, Mo.; 28.4 mg of heparin sodium in 500 ml of distilled water), andthe mixture was incubated at 37° C. for 1 hour. The upper layercontaining the leukocytes was collected by centrifugation, and hypotoniclysis of the remaining erythrocytes was accomplished by suspension ofthe cell pellet in 1% (w/v) NH₄Cl. The leukocyte population wassubsequently washed in RPMI with 15% fetal bovine serum. Trypan bluestaining and counting in a hemocytometer were used to determine theconcentration of live leukocytes, and the final leukocyte concentrationwas adjusted to 2×10⁷ cells/ml. The phagocytosis assay was performed induplicate with or without 100 μl of leukocyte suspension added to 100 μlof bacteria (concentration adjusted spectrophotometrically to 2×10⁷ perml and confirmed by viable counts), 100 μl of anti-enterococci humanIgG1 diluted in RPMI, and 100 μl of baby rabbit complement. The reactionmixture was incubated on a rotor rack at 37° C. for 90 minutes; sampleswere taken at time 0 and after 90 minutes, diluted in 1% ProteosePeptone (Difco Laboratories, Detroit, Mich.), and plated onto trypticsoy agar plates. The killing activity (%) of the antibodies wascalculated as the mean number of CFU surviving in the sample containingleukocytes subtracted from the mean number of CFU surviving in thesample without leukocytes, divided by the latter, and amplified by 100.Four concentrations of the anti-enterococci human IgG1 were tested(2500, 250, 25, 2.5 ng/ml) in two independent experiments. Ordinalregression analysis applying the probit model was used to calculate theconcentrations required for 50% killing of bacteria in the assay (seeTable 14).

Example 8 In Vivo Activity of Enterococcal Specific IgGs in a MurineSepsis Model

A murine sepsis model of enterococcus (see Hufnagel et al. 2004) wasused to quantify activity of anti-enterococci human IgG1 in clearing theenterococcal clinical isolate 12030 from the bloodstream. The purifiedIgG1 molecules CR5159, CR5187, CR6016, CR6043, CR6049, CR6071, CR6089,and CR6241 demonstrated to have in vitro killing activity againstEnterococcus and one negative control IgG1 having no killing activityagainst Enterococcus were prepared as described above and were injectedi.p. (0.5-1 ml in PBS) into groups of eight BALB/c mice at a dose of 15mg/kg, with the exception of CR6016 and CR6241 which were injected at adose of 7.5 mg/kg. In addition, one group of mice was injected with PBS.After 24 hours animals were inoculated i.v. with 6×10⁸ CFU ofEnterococcus strain 12030. Four hours after the bacterial challenge micereceived a second i.p. injection of CR5159, CR5187, CR6016, CR6043,CR6049, CR6071, CR6089, and CR6241 at the same dose. Three days aftersystemic infection animals were euthanized and ˜0.5 ml of bloodcollected by cardiac puncture. Blood samples were culturedquantitatively on enterococcal selective agar medium; 100 μl of blooddiluted in 900 μl of THB was spread out onto plates in duplicate. Afterovernight incubation the number of CFU was read off the plate andmultiplied by 10 to give the CFU/ml of blood. This value is directlyrelated to the amount of circulating bacteria at the time of sacrifice.

The primary endpoint in this model is CFU of Enterococcus in the blood 3days after inoculation. As shown in FIG. 1 all of the animals thatreceived PBS or control IgG1 had >10² CFU/ml of Enterococcus in theirblood after 3 days and the median was ˜10³ CFU/ml. In contrast, all ofthe groups that received anti-enterococcal antibodies contained animalswith <10² CFU/ml of Enterococcus in the blood. In addition, in all butone case, CR5187, the median was one log below that of the controls. Oneantibody, CR6089, had a median below the level of sensitivity in theassay (10 CFU/ml) and in 6 out of 8 animals there was no detectablebacteria in the blood. CR6016 and CR6241 that were used at a lower dosestill had medians close to 10 CFU/ml of blood indicating that they areof high potency. Non-parametric analysis of variance (Kruskal-Wallis)established that the differences were highly significant (p<0.001).Pairwise comparisons were performed between the test IgG1 and negativecontrol IgG1 using the Mann-Whitney test with the Bonferroni correction.Antibodies CR5159, CR5187, CR6043, CR6049, CR6089, and CR6241 were allsignificantly different (p<0.05) to the control antibody, while themedian difference of the antibodies CR6043 and CR6071 did not reachsignificance when compared to the control antibody.

Example 9 IgG1 Competition Assay

To establish whether antibodies in the panel competed for binding to thesame target a competition ELISA was developed. The enterococcal strain12030 was streaked onto a blood agar plate and incubated overnight at37° C. Colonies were scraped from the plate using 5 ml of a 50 mMcarbonate buffer (8 volumes of 0.2 M Na2CO3, 17 volumes of 0.2 M NaHCO3and 75 volumes of distilled water) and centrifuged for 3 minutes at 4000rpm. The pellet obtained was resuspended in 500 μl of carbonate buffer,centrifuged again and the pellet was resuspended in 500 μl carbonatebuffer. Cell density was determined by measuring OD600 of a dilutionseries of the bacteria.

The enterococcus strain was diluted to a density of 5×10⁹ cells/ml and100 μl (5×10⁸ cells) per well was coated overnight at 4° C. onNunc-Immuno Maxisorp™ F96 plates. After incubation, the wells werewashed three times with PBS and blocked for 1 hour at room temperaturewith 300 μl 2% (v/v) ELK in PBS per well. In separate tubes 25 μl ofeach scFv-phage maxiprep (produced as above) diluted to sub-saturatinglevels (as determined by ELISA above) was mixed with 25 μl blockingbuffer (4% (v/v) ELK in PBS) and 50 μl of IgG1 supernatant diluted to 10μg/ml in PBS. The mixture was incubated for 20 minutes on ice. Afterremoving the blocking solution from the wells, 100 μl of the mixture wasadded to each well and incubated for 1 hour at room temperature. Next,the wells were washed three times with PBS/0.01% (v/v) Tween® and oncewith PBS. After washing, 100 μl of anti-M13 HRP (1:5000 in 2% (v/v) ELKin PBS) was added per well and incubated for 60 minutes at roomtemperature. The wells were washed again and staining was visualized byadding 100 μl OPD-solution to each well. The visualization reaction wasstopped after 5-10 minutes by adding 50 μl 1 M H₂SO₄ to each well andthe OD was measured at 492 nm. The experiment was repeated twice withthe entire panel of antibodies and the control IgG1 CR4374. The resultsshowed that the antibodies could be divided into several distinctgroups. Group A consisted of CR6089 and CR6092; Group B consisted ofCR5157, CR5187, CR6043, CR6049, CR6388, CR6389, CR6396, CR6402, CR6409,CR6421, and CR6429; and Group C consisted of CR5159, CR5166, CR6050,CR6077, CR6078, CR6086, and CR6191 and the rest of the antibodiesCR5140, CR5179, CR6016, CR6071, CR6079, CR6087, CR6195, CR6198, CR6241,CR6242, CR6246, CR6252, CR6415, and CR6432 did not compete with anyother antibody for binding.

Example 10 In Vitro Opsonic Phagocytic Activity of Anti-EnterococcalIgG1 Molecules Against Different E. faecalis, E. faecium and S. aureusStrains Measured by Opsonophagocytic Killing Assay

To determine the breadth of killing activity of the anti-enterococcalmonoclonal antibody panel, purified batches of IgG1 made as describedabove were assayed for killing activity in the opsonophagocytic killingassay described above. An additional E. faecalis strain, Type 2; twodifferent E. faecium clinical isolates, 740220 and 838970; and the S.aureus clinical isolate 502 were tested. Eighteen antibodies were chosenfrom the original panel of 34 based on non-competing binding capacityand potency in the opsonophagocytic killing assay. As shown in Table 15,the chosen panel showed killing activity against the E. faecium strainsat two concentrations, 2.5 and 0.025 μg/ml, although the activity ofCR5140, CR6016 and CR6078 was lower than 20% against strain 838970 atthe highest concentration. All but one antibody had measurable activityagainst E. faecalis strain Type 2, although 11 out of 18 antibodies hadless than 25% killing activity at the highest concentration tested.Surprisingly, all antibodies of the panel had killing activity againstthe S. aureus strain 502, indicating that the antibodies recognizebroadly cross-reactive targets. We tested whether any of the antibodiesbind to lipoteichoic acid (LTA) of S. aureus, and none of theseantibodies appeared to do so. Three of the antibodies (CR6252, CR6415and CR6421) were tested for opsonic phagocytic killing activity againstanother Staphylococcus aureus strain (Newman), and against a S.epidermidis strain (RP62A), and all three antibodies tested showedkilling activity against these different Staphylococcus species andstrains.

TABLE 1 Human lambda chain variable region primers (sense). PrimerPrimer name nucleotide sequence SEQ ID NO HuVL1A-Back5′-CAGTCTGTGCTGACTCA SEQ ID NO: 130 GCCACC-3′ HuVL1B-Back5′-CAGTCTGTGYTGACGCA SEQ ID NO: 131 GCCGCC-3′ HuVL1C-Back5′-CAGTCTGTCGTGACGCA SEQ ID NO: 132 GCCGCC-3′ HuVL2B-Back5′-CAGTCTGCCCTGACTCA SEQ ID NO: 133 GCC-3′ HuVL3A-Back5′-TCCTATGWGCTGACTCA SEQ ID NO: 134 GCCACC-3′ HuVL3B-Back5′-TCTTCTGAGCTGACTCA SEQ ID NO: 135 GGACCC-3′ HuVL4B-Back5′-CAGCYTGTGCTGACTCA SEQ ID NO: 136 ATC-3′ HuVL5-Back5′-CAGGCTGTGCTGACTCA SEQ ID NO: 137 GCCGTC-3′ HuVL6-Back5′-AATTTTATGCTGACTCA SEQ ID NO: 138 GCCCCA-3′ HuVL7/8-Back5′-CAGRCTGTGGTGACYCA SEQ ID NO: 139 GGAGCC-3′ HuVL9-Back5′-CWGCCTGTGCTGACTCA SEQ ID NO: 140 GCCMCC-3′ HuVL10-Back5′-CAGGCAGGGCTGACTCA SEQ ID NO: 141 G-3′

TABLE 2 Human kappa chain variable region primers (sense). PrimerPrimer name nucleotide sequence SEQ ID NO HuVK1B-Back 5′-GACATCCAGWTGACCSEQ ID NO: 142 CAGTCTCC-3′ HuVK2-Back 5′-GATGTTGTGATGACT SEQ ID NO: 143CAGTCTCC-3′ HuVK2B2 5′-GATATTGTGATGACC SEQ ID NO: 144 CAGACTCC-3′HuVK3B-Back 5′-GAAATTGTGWTGACR SEQ ID NO: 145 CAGTCTCC-3′ HuVK5-Back5′-GAAACGACACTCACG SEQ ID NO: 146 CAGTCTCC-3′ HuVK6-Back5′-GAAATTGTGCTGACT SEQ ID NO: 147 CAGTCTCC-3′

TABLE 3 Human kappa chain variable region primers extended with SalIrestriction sites (sense), human kappa chain J-region primers extendedwith NotI restriction sites (anti-sense), human lambda chain variable regionprimers extended with SalI restriction sites (sense) and human lambda chainJ-region primers extended with NotI restriction sites (anti-sense).Primer name Primer nucleotide sequence SEQ ID NO HuVK1B-Back-SAL5′-TGAGCACACAGGTCGACGGACATCCAGWTGACCCAGTCTCC-3′ SEQ ID NO: 148HuVK2-Back-SAL 5′-TGAGCACACAGGTCGACGGATGTTGTGATGACTCAGTCTCC-3′SEQ ID NO: 149 HuVK2B2-SAL5′-TGAGCACACAGGTCGACGGATATTGTGATGACCCAGACTCC-3′ SEQ ID NO: 150HuVK3B-Back-SAL 5′-TGAGCACACAGGTCGACGGAAATTGTGWTGACRCAGTCTCC-3′SEQ ID NO: 151 HuVK5-Back-SAL5′-TGAGCACACAGGTCGACGGAAACGACACTCACGCAGTCTCC-3′ SEQ ID NO: 152HuVK6-Back-SAL 5′-TGAGCACACAGGTCGACGGAAATTGTGCTGACTCAGTCTCC-3′SEQ ID NO: 153 HuJK1-FOR-NOT5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATTTCCACCTTGGTCCC-3′ SEQ ID NO: 154HuJK2-FOR-NOT 5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATCTCCAGCTTGGTCCC-3′SEQ ID NO: 155 HuJK3-FOR-NOT5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATATCCACTTTGGTCCC-3′ SEQ ID NO: 156HuJK4-FOR-NOT 5′-GAGTCATTCTCGACT TGCGGCCGACGTTTGATCTCCACCTTGGTCCC-3′SEQ ID NO: 157 HuJK5-FOR-NOT5′-GAGTCATTCTCGACTTGCGGCCGCACGTTTAATCTCCAGTCGTGTCCC-3′ SEQ ID NO: 158HuVL1A-Back-SAL 5′-TGAGCACACAGGTCGACGCAGTCTGTGCTGACTCAGCCACC-3′SEQ ID NO: 159 HuVL1B-Back-SAL5′-TGAGCACACAGGTCGACGCAGTCTGTGYTGACGCAGCCGCC-3′ SEQ ID NO: 160HuVL1C-Back-SAL 5′-TGAGCACACAGGTCGACGCAGTCTGTCGTGACGCAGCCGCC-3′SEQ ID NO: 161 HuVL2B-Back-SAL5′-TGAGCACACAGGTCGACGCAGTCTGCCCTGACTCAGCC-3′ SEQ ID NO: 162HuVL3A-Back-SAL 5′-TGAGCACACAGGTCGACGTCCTATGWGCTGACTCAGCCACC-3′SEQ ID NO: 163 HuVL3B-Back-SAL5′-TGAGCACACAGGTCGACGTCTTCTGAGCTGACTCAGGACCC-3′ SEQ ID NO: 164HuVL4B-Back-SAL 5′-TGAGCACACAGGTCGACGCAGCYTGTGCTGACTCAATC-3′SEQ ID NO: 165 HuVL5-Back-SAL5′-TGAGCACACAGGTCGACGCAGGCTGTGCTGACTCAGCCGTC-3′ SEQ ID NO: 166HuVL6-Back-SAL 5′-TGAGCACACAGGTCGACGAATTTTATGCTGACTCAGCCCCA-3′SEQ ID NO: 167 HuVL7/8-Back-SAL5′-TGAGCACACAGGTCGACGCAGRCTGTGGTGACYCAGGAGCC-3′ SEQ ID NO: 168HuVL9-Back-SAL 5′-TGAGCACACAGGTCGACGCWGCCTGTGCTGACTCAGCCMCC-3′SEQ ID NO: 169 HuVL10-Back-SAL5′-TGAGCACACAGGTCGACGCAGGCAGGGCTGACTCAG-3′ SEQ ID NO: 170 HuJL1-FOR-NOT5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTGACCTTGGTCCC-3′ SEQ ID NO: 171HuJL2/3-FOR-NOT 5′-GAGTCATTCTCGACTTGCGGCCGCACCTAGGACGGTCAGCTTGGTCCC-3′SEQ ID NO: 172 HuJL7-FOR-NOT5′-GAGTCATTCTCGACTTGCGGCCGCACCGAGGACGGTCAGCTGGGTGCC-3′ SEQ ID NO: 173

TABLE 4 Percentage of the different light chain products in the finalmixture, based on concentrations determined by agarose gel analysis.Sense primer Antisense primer Product Percentage HuVL1A-Back-SAL +HuJL1-FOR-NOT L1J1 4.20% HuVL1B-Back-SAL + HuJL2/3-FOR-NOT L1J2 8.40%HuVL1C-Back-SAL HuJL7-FOR-NOT L1J3 1.40% HuVL2B-Back-SAL HuJL1-FOR-NOTL2J1 3.00% HuJL2/3-FOR-NOT L2J2 6.00% HuJL7-FOR-NOT L2J3 1.00%HuVL3A-Back-SAL HuJL1-FOR-NOT L3J1 3.00% HuJL2/3-FOR-NOT L3J2 6.00%HuJL7-FOR-NOT L3J3 1.00% HuVL3B-Back-SAL HuJL1-FOR-NOT L4J1 0.30%HuJL2/3-FOR-NOT L4J2 0.60% HuJL7-FOR-NOT L4J3 0.10% HuVL4B-Back-SALHuJL1-FOR-NOT L5J1 0.30% HuJL2/3-FOR-NOT L5J2 0.60% HuJL7-FOR-NOT L5J30.10% HuVL5-Back-SAL HuJL1-FOR-NOT L6J1 0.30% HuJL2/3-FOR-NOT L6J2 0.60%HuJL7-FOR-NOT L6J3 0.10% HuVL6-Back-SAL HuJL1-FOR-NOT L7J1 0.30%HuJL2/3-FOR-NOT L7J2 0.60% HuJL7-FOR-NOT L7J3 0.10% HuVL7/8-Back-SALHuJL1-FOR-NOT L8J1 0.30% HuJL2/3-FOR-NOT L8J2 0.60% HuJL7-FOR-NOT L8J30.10% HuVL9-Back-SAL + HuJL1-FOR-NOT L9J1 0.30% HuVL10-Back-SALHuJL2/3-FOR-NOT L9J2 0.60% HuJL7-FOR-NOT L9J3 0.10% HuVK1B-Back-SALHuJK1-FOR-NOT K1J1 7.50% HuJK2-FOR-NOT K1J2 7.50% HuJK3-FOR-NOT K1J33.00% HuJK4-FOR-NOT K1J4 7.50% HuJK5-FOR-NOT K1J5 4.50% HuVK2-Back-SALHuJK1-FOR-NOT K2J1 1.00% HuJK2-FOR-NOT K2J2 1.00% HuJK3-FOR-NOT K2J30.40% HuJK4-FOR-NOT K2J4 1.00% HuJK5-FOR-NOT K2J5 0.60% HuVK2B2-SALHuJK1-FOR-NOT K3J1 0.25% HuJK2-FOR-NOT K3J2 0.25% HuJK3-FOR-NOT K3J30.10% HuJK4-FOR-NOT K3J4 0.25% HuJK5-FOR-NOT K3J5 0.15% HuVK3B-Back-SALHuJK1-FOR-NOT K4J1 4.75% HuJK2-FOR-NOT K4J2 4.75% HuJK3-FOR-NOT K4J31.90% HuJK4-FOR-NOT K4J4 4.75% HuJK5-FOR-NOT K4J5 2.85% HuVK5-Back-SALHuJK1-FOR-NOT K5J1 0.25% HuJK2-FOR-NOT K5J2 0.25% HuJK3-FOR-NOT K5J30.10% HuJK4-FOR-NOT K5J4 0.25% HuJK5-FOR-NOT K5J5 0.15% HuVK6-Back-SALHuJK1-FOR-NOT K6J1 1.25% HuJK2-FOR-NOT K6J2 1.25% HuJK3-FOR-NOT K6J30.50% HuJK4-FOR-NOT K6J4 1.25% HuJK5-FOR-NOT K6J5 0.75%

TABLE 5 Human IgG heavy chain variable region primers (sense). PrimerPrimer name nucleotide sequence SEQ ID NO HuVH1B/7A-Back5′-CAGRTGCAGCTGGTGCA SEQ ID NO: 174 RTCTGG-3′ HuVH1C-Back5′-SAGGTCCAGCTGGTRCA SEQ ID NO: 175 GTCTGG-3′ HuVH2B-Back5′-CAGRTCACCTTGAAGGA SEQ ID NO: 176 GTCTGG-3′ HuVH3A-Back5′-GAGGTGCAGCTGGTGGA SEQ ID NO: 177 G-3′ HuVH3C-Back5′-GAGGTGCAGCTGGTGGA SEQ ID NO: 178 GWCYGG-3′ HuVH4B-Back5′-CAGGTGCAGCTACAGCA SEQ ID NO: 179 GTGGGG-3′ HuVH4C-Back5′-CAGSTGCAGCTGCAGGA SEQ ID NO: 180 GTCSGG-3′ HuVH6A-Back5′-CAGGTACAGCTGCAGCA SEQ ID NO: 181 GTCAGG-3′

TABLE 6Human IgG heavy chain variable region primers extended with SfiI/NcoIrestriction sites (sense) and human IgG heavy chain J-regionprimers extended with XhoI/BstEII restriction sites (anti-sense).Primer name Primer nucleotide sequence SEQ ID NO HuVH1B/7A-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 182CAGRTGCAGCTGGTGCARTCTGG-3′ HuVH1C-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 183SAGGTCCAGCTGGTRCAGTCTGG-3′ HuVH2B-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 184CAGRTCACCTTGAAGGAGTCTGG-3′ HuVH3A-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 185GAGGTGCAGCTGGTGGAG-3′ HuVH3C-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 186GAGGTGCAGCTGGTGGAGWCYGG-3′ HuVH4B-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 187CAGGTGCAGCTACAGCAGTGGGG-3′ HuVH4C-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 188CAGSTGCAGCTGCAGGAGTCSGG-3′ HuVH6A-Back-Sfi5′-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCC SEQ ID NO: 189CAGGTACAGCTGCAGCAGTCAGG-3′ HuJH1/2-FOR-XhoIB5′-GAGTCATTCTCGACTCGAGACRGTGACCAGGGT SEQ ID NO: 190 GCC-3′ HuJH3-FOR-Xho5′-GAGTCATTCTCGACTCGAGACGGTGACCATTGT SEQ ID NO: 191 CCC-3′HuJH4/5-FOR-Xho 5′-GAGTCATTCTCGACTCGAGACGGTGACCAGGGT SEQ ID NO: 192TCC-3′ HuJH6-FOR-Xho 5′-GAGTCATTCTCGACTCGAGACGGTGACCGTGGT SEQ ID NO: 193CCC-3′

TABLE 7 Percentage of the different heavy chain products in the finalmixture. Sense primer Antisense primer Product PercentageHuVH1B/7A-Back-Sfi + HuJH1/2-FOR-XhoIB H1J1 2.5% HuVH1C-Back-SfiHuJH3-FOR-Xho H1J2 2.5% HuJH4/5-FOR-Xho H1J3 15.0% HuJH6-FOR-Xho H1J45.0% HuVH2B-Back-Sfi HuJH1/2-FOR-XhoIB H2J1 0.2% HuJH3-FOR-Xho H2J2 0.2%HuJH4/5-FOR-Xho H2J3 1.2% HuJH6-FOR-Xho H2J4 0.4% HuVH3A-Back-SfiHuJH1/2-FOR-XhoIB H3J1 2.5% HuJH3-FOR-Xho H3J2 2.5% HuJH4/5-FOR-Xho H3J315.0% HuJH6-FOR-Xho H3J4 5.0% HuVH3C-Back-Sfi HuJH1/2-FOR-XhoIB H4J12.5% HuJH3-FOR-Xho H4J2 2.5% HuJH4/5-FOR-Xho H4J3 15.0% HuJH6-FOR-XhoH4J4 5.0% HuVH4B-Back-Sfi HuJH1/2-FOR-XhoIB H5J1 0.2% HuJH3-FOR-Xho H5J20.2% HuJH4/5-FOR-Xho H5J3 1.2% HuJH6-FOR-Xho H5J4 0.4% HuVH4C-Back-SfiHuJH1/2-FOR-XhoIB H6J1 2.0% HuJH3-FOR-Xho H6J2 2.0% HuJH4/5-FOR-Xho H6J312.0% HuJH6-FOR-Xho H6J4 4.0% HuVH6A-Back-Sfi HuJH1/2-FOR-XhoIB H7J10.1% HuJH3-FOR-Xho H7J2 0.1% HuJH4/5-FOR-Xho H7J3 0.6% HuJH6-FOR-XhoH7J4 0.2%

TABLE 8 Enterococcal strains used for selection and screening ofanti-enterococcal single-chain (scFv) phage antibodies. Strain Source E.faecalis 12030 Veterans Administration Hospital, Cleveland, Ohio E.faecalis T2 Prototype Japanese strain E. faecalis 6814 Brigham andWomen's Hospital, Boston, Massachusetts E. faecalis B8610A Brigham andWomen's Hospital, Boston, Massachusetts E. faecium 740220 Brigham andWomen's Hospital, Boston, Massachusetts E. faecium B210860 Brigham andWomen's Hospital, Boston, Massachusetts

TABLE 9 Enterococcal specific binding activity of single-chain (scFv)phage antibodies as measured by ELISA. Enterococcus Control strainsantigens Name phage (OD492 nm) (OD492 nm) antibody 12030 T2 BSA ELKSC05-140 1.094 ND 0.226 0.152 SC05-157 0.787 ND 0.058 0.106 SC05-1590.612 ND 0.060 0.089 SC05-166 0.954 ND 0.104 0.099 SC05-179 0.804 ND0.045 0.047 SC05-187 0.835 1.043 0.055 0.055 SC06-016 0.842 ND 0.0440.041 SC06-043 0.705 ND 0.045 0.042 SC06-049 0.241 ND 0.042 0.043SC06-050 0.410 ND 0.043 0.043 SC06-071 0.703 0.746 0.043 0.042 SC06-0770.577 1.005 0.044 0.060 SC06-078 0.596 1.040 0.073 0.044 SC06-079 0.6630.953 0.048 0.041 SC06-086 0.587 ND 0.062 0.053 SC06-087 0.553 ND 0.0440.060 SC06-089 0.613 ND 0.042 0.063 SC06-092 0.624 ND 0.047 0.050SC06-191 0.456 0.498 0.044 0.039 SC06-195 0.661 0.789 0.046 0.043SC06-198 0.999 1.169 0.049 0.044 SC06-241 1.107 0.122 0.052 0.045SC06-242 0.814 0.085 0.043 0.043 SC06-246 0.588 0.636 0.042 0.040SC06-252 0.638 0.304 0.044 0.039 SC06-388 1.006 1.301 ND 0.040 SC06-3891.337 1.743 ND 0.038 SC06-396 0.689 1.166 ND 0.067 SC06-402 1.538 1.905ND 0.126 SC06-409 0.876 1.339 0.055 0.051 SC06-415 0.889 1.565 0.0440.049 SC06-421 3.150 3.270 0.607 0.133 SC06-429 1.101 2.453 0.068 0.043SC06-432 0.807 2.401 0.059 0.044 Average neg. ctrl 0.12  0.15  0.07 0.06  ND means not determined

TABLE 10 Data of the Enterococcus specific single-chain Fvs. SEQ ID NOSEQ ID NO of of nucl. amino acid Name scFv sequence sequence* VH-locusVL-locus SC05-140 350 351 Vh3 (3-33) Vl 3 (3h-V2-14) (Vh 1-121; Vl138-243) SC05-157 352 353 Vh5 (5-51) Vl 1 (1c-V1-16) (Vh 1-121; Vl138-247) SC05-159 61  62 VH1 (1-f) Vl 1 (1c-V1-16) (Vh 1-123; Vl140-249) SC05-166 63  64 VH1 (1-18) Vl 6 (6a-V1-22) (Vh 1-133; Vl150-259) SC05-179 354 355 Vh3 (3-11) Vl 2 (2e-V1-03) (Vh 1-117; Vl134-244) SC05-187 65  66 VH5 (5-51) Vl 7 (7a-V3-02) (Vh 1-121; Vl138-246) SC06-016 67  68 VH1 (1-18) Vk I (L5-DPK5) (Vh 1-118; Vl135-241) SC06-043 69  70 VH5 (5-51) Vl 2 (2c-V1-02) (Vh 1-123; Vl140-249) SC06-049 71  72 VH5 (5-51) Vl 2 (2a2-V1-04) (Vh 1-120; Vl137-246) SC06-050 356 357 Vh1 (1-18) Vk I (L8-DPK8) (Vh 1-126; Vl143-249) SC06-071 73  74 VH3 (3-33) Vl 2 (2a2-V1-04 (Vh 1-122; Vl139-248) SC06-077 358 359 Vh1 (1-69) Vk IV (B3-DPK24) (Vh 1-119; Vl136-248) SC06-078 75  76 VH1 (1-69) Vl 2 (2a2-V1-04) (Vh 1-119; Vl136-245) SC06-079 360 361 Vh3 (3-23) Vl 1 (1g-V1-17) (Vh 1-116; Vl133-242) SC06-086 362 363 Vh1 (1-69) Vk I (O12/O2- (Vh 1-120; DPK9) Vl137-243) SC06-087 206 207 Vh3 (3-21) Vl 2 (2a2-V1-04) (Vh 1-122; Vl139-249) SC06-089 208 209 Vh3 (3-48) Vl 3 (3h-V2-14) (Vh 1-123; Vl140-247) SC06-092 364 365 Vh3 (3-49) Vl 2 (2a2-V1-04) (Vh 1-121; Vl138-248) SC06-191 366 367 Vh3 (3-33) Vk I (L12) (Vh 1-120; VI 137-243)SC06-195 368 369 Vh3 (3-33) Vl 1 (1g-V1-17) (Vh 1-115; Vl 132-241)SC06-198 370 371 Vh4 (4-b) Vl 1 (1e-V1-13) (Vh 1-116; Vl 133-243)SC06-241 77  78 VH3 (3-30.3) Vk I (L5-DPK5) (Vh 1-118; Vl 135-241)SC06-242 372 373 Vh4 (4-59) Vl 3 (3h-V2-14) (Vh 1-115; Vl 132-237)SC06-246 374 375 Vh3 (3-53) Vl 3 (3h-V2-14) (Vh 1-121; Vl 138-245)SC06-252 79  80 VH3 (3-23) Vl 1 (1c-V1-16) (Vh 1-115; Vl 132-241)SC06-388 376 377 Vh5 (5-51) Vl 2 (2c-V1-02) (Vh 1-119; Vl 136-245)SC06-389 378 379 Vh5 (5-51) Vl 3 (31-V2-13) (Vh 1-121; Vl 138-245)SC06-396 380 381 Vh5 (5-51) Vk III (A27- (Vh 1-121; DPK22) Vl 138-245)SC06-402 382 383 Vh5 (5-51) Vl 2 (2e-V1-03) (Vh 1-127; Vl 144-255)SC06-409 384 385 Vh5 (5-51) Vl 2 (2a2-V1-04) (Vh 1-122; V1 139-249)SC06-415 386 387 Vh3 (3-09) Vl 2 (2c-V1-02) (Vh 1-116; Vl 133-242)SC06-421 388 389 Vh5 (5-51) Vl 2 (2c-V1-02) (Vh 1-120; Vl 137-246)SC06-429 390 391 Vh5 (5-51) Vk II (A19/A03- (Vh 1-121; DPK15) Vl138-249) SC06-432 392 393 Vh4 (4-31) Vl 1 (1e-V1-13) (Vh 1-120; Vl137-246) *between brackets the amino acids making up the heavy chainvariable region (VH) and the light chain variable region (VL) is shown

TABLE 11 Data of the CDR regions of the Enterococcus specificsingle-chain Fvs. HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Name scFv (SEQ IDNO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)SC05-140 218 219 220 221 222 223 SC05-157 224 225 226 227 228 229SC05-159 1 2 3 4 5 6 SC05-166 7 8 9 10 11 12 SC05-179 230 231 232 233234 235 SC05-187 13 14 15 16 17 18 SC06-016 19 20 21 22 23 24 SC06-04325 26 27 28 29 30 SC06-049 31 32 33 34 35 36 SC06-050 236 237 238 239240 241 SC06-071 37 38 39 40 41 42 SC06-077 242 243 244 245 246 247SC06-078 43 44 45 46 47 48 SC06-079 248 249 250 251 252 253 SC06-086 254255 256 257 258 259 SC06-087 194 195 196 197 198 199 SC06-089 200 201202 203 204 205 SC06-092 260 261 262 263 264 265 SC06-191 266 267 268269 270 271 SC06-195 272 273 274 275 276 277 SC06-198 278 279 280 281282 283 SC06-241 49 50 51 52 53 54 SC06-242 284 285 286 287 288 289SC06-246 290 291 292 293 294 295 SC06-252 55 56 57 58 59 60 SC06-388 296297 298 299 300 301 SC06-389 302 303 304 305 306 307 SC06-396 308 309310 311 312 313 SC06-402 314 315 316 317 318 319 SC06-409 320 321 322323 324 325 SC06-415 326 327 328 329 330 331 SC06-421 332 333 334 335336 337 SC06-429 338 339 340 341 342 343 SC06-432 344 345 346 347 348349

TABLE 12 Data of the Enterococcus specific IgGs. SEQ ID NO of SEQ ID NOof amino SEQ ID NO of SEQ ID NO of amino Name nucl. sequence acidsequence* heavy nucl. sequence acid sequence* light IgG heavy chainchain light chain chain CR5140 394 395 438 439 (Vh 1-121) (Vl 1-106)CR5157 396 397 440 441 (Vh 1-121) (Vl 1-110) CR5159 81  82 101 102 (Vh1-123) (Vl 1-110) CR5166 83  84 103 104 (Vh 1-133) (Vl 1-110) CR5179 398399 442 443 (Vh 1-117) (Vl 1-111) CR5187 85  86 105 106 (Vh 1-121) (Vl1-109) CR6016 87  88 107 108 (Vh 1-118) (Vl 1-107) CR6043 89  90 109 110(Vh 1-123) (Vl 1-110) CR6049 91  92 111 112 (Vh 1-120) (Vl 1-110) CR6050400 401 444 445 (Vh 1-126) (Vl 1-107) CR6071 93  94 113 114 (Vh 1-122)(Vl 1-110) CR6077 402 403 446 447 (Vh 1-119) (Vl 1-133) CR6078 95  96115 116 (Vh 1-119) (Vl 1-110) CR6079 404 405 448 449 (Vh 1-116) (Vl1-110) CR6086 406 407 450 451 (Vh 1-120) (Vl 1-107) CR6087 210 211 214215 (Vh 1-122) (Vl 1-111) CR6089 212 213 216 217 (Vh 1-123) (Vl 1-108)CR6092 408 409 452 453 (Vh 1-121) (Vl 1-111) CR6191 410 411 454 455 (Vh1-120) (Vl 1-107) CR6195 412 413 456 457 (Vh 1-115) (Vl 1-110) CR6198414 415 458 459 (Vh 1-116) (Vl 1-111) CR6241 97  98 117 118 (Vh 1-118)(Vl 1-107) CR6242 416 417 460 461 (Vh 1-115) (Vl 1-106) CR6246 418 419462 463 (Vh 1-121) (Vl 1-108) CR6252 99 100 119 120 (Vh 1-115) (Vl1-110) CR6388 420 421 464 465 (Vh 1-119) (Vl 1-110) CR6389 422 423 466467 (Vh 1-121) (Vl 1-108) CR6396 424 425 468 469 (Vh 1-121) (Vl 1-108)CR6402 426 427 470 471 (Vh 1-127) (Vl 1-112) CR6409 428 429 472 473 (Vh1-122) (Vl 1-111) CR6415 430 431 474 475 (Vh 1-116) (Vl 1-110) CR6421432 433 476 477 (Vh 1-120) (Vl 1-110) CR6429 434 435 478 479 (Vh 1-121)(Vl 1-112) CR6432 436 437 480 481 (Vh 1-120) (Vl 1-110) *betweenbrackets the amino acids making up the heavy chain variable region (VH)and the light chain variable region (VL) is shown

TABLE 13 Specific binding activity against different strains ofEnterococcus faecalis and Enterococcus faecium by human IgG1 antibodiesas measured by ELISA. Antibody Enterococcal strains (OD492 nm) Name12030 T2 6814 B8610A 740220* B210860* CR5140 3.084 0.185 0.121 1.7690.185 0.123 CR5157 0.225 0.358 0.215 0.282 0.199 0.086 CR5159 0.3830.441 0.265 0.134 0.114 0.077 CR5166 0.533 1.387 0.444 0.140 0.170 0.101CR5179 0.250 1.206 0.285 1.546 0.131 0.091 CR5187 0.869 1.267 0.9391.269 0.725 0.296 CR6016 0.281 0.622 0.243 0.134 0.126 0.084 CR60430.232 0.326 0.203 0.274 0.196 0.101 CR6049 0.779 1.258 1.123 0.992 0.5090.251 CR6050 0.291 0.739 0.218 0.117 0.138 0.092 CR6071 1.452 0.3910.699 0.629 0.109 0.081 CR6077 0.739 1.774 0.436 0.137 0.137 0.086CR6078 0.482 1.457 0.336 0.143 0.114 0.082 CR6079 0.751 0.597 0.2931.160 0.186 0.114 CR6086 0.583 1.554 0.335 0.118 0.116 0.080 CR60871.085 1.414 0.098 0.135 0.182 0.091 CR6089 2.164 0.309 1.127 0.822 0.1180.085 CR6092 2.779 1.204 1.989 1.599 0.113 0.088 CR6191 0.868 1.6390.475 0.100 0.063 0.043 CR6195 0.304 1.652 0.084 2.219 0.051 0.042CR6198 1.151 2.854 0.532 2.849 0.071 0.039 CR6241 0.814 0.091 0.0430.072 0.060 0.037 CR6242 0.356 0.102 0.047 0.075 0.079 0.038 CR62460.207 0.290 0.047 0.083 0.131 0.049 CR6252 0.583 0.370 0.045 0.076 0.6900.052 CR6388 0.165 0.180 0.139 0.157 0.207 0.116 CR6389 0.562 0.1970.122 0.182 0.168 0.320 CR6396 0.427 0.640 0.342 0.500 0.456 0.312CR6402 0.428 0.391 0.236 0.447 0.292 0.270 CR6409 0.120 0.155 0.1130.145 0.169 0.124 CR6415 2.284 1.910 0.122 0.108 1.119 0.195 CR64210.693 0.803 0.511 0.822 0.438 0.368 CR6429 0.302 0.437 0.190 0.403 0.3470.185 CR6432 0.358 0.364 0.322 0.500 0.216 0.406 Average neg. 0.11 0.130.09 0.13 0.12 0.07 ctrl

TABLE 14 In vitro opsonophagocytic killing activity against Enterococcusfaecalis strain 12030 by human IgG1 antibodies. Antibody AntibodyConcentrations (ng/ml) giving Name 50% bacterial killing CR5140 NDCR5157 20.7 CR5159 130 CR5166 27.8 CR5179 312 CR5187 295 CR6016 2.20CR6043 8.94 CR6049 3794 CR6050 5.82 CR6071 12.4 CR6077 54.7 CR6078 10.5CR6079 >10000 CR6086 10.8 CR6087 21.2 CR6089 3.67 CR6092 >10000 CR6191178 CR6195 >10000 CR6198 4787 CR6241 0.613 CR6242 ND CR6246 >10000CR6252 29.2 CR6388 0.64 CR6389 0.33 CR6396 4.71 CR6402 1.00 CR6409 36.6CR6415 ND CR6421 21.6 CR6429 1.2 CR6432 >10000 ND means not determined

TABLE 15 Killing activity of IgG1 antibodies as measured byopsonophagocytic killing assay. Mean enterococcal and staphylococcalkilling activity (%) Strain Type 2 740220 838970 502 IgG1 [ng/ml]antibody 2500 25 2500 25 2500 25 2500 25 CR5140 14.9 7.7 72.2 44.1 11.92.3 66.4 44.9 CR5157 2.3 4.0 64.8 14.5 27.7 9.7 48.7 27.0 CR6016 15.74.6 66.9 17.9 3.2 1.7 59.0 32.3 CR6043 30.0 16.1 63.6 15.7 21.1 2.8 50.521.3 CR6050 7.5 5.8 49.4 18.8 33.1 8.1 59.0 28.2 CR6078 43.2 24.9 60.425.6 4.6 1.5 39.2 12.8 CR6087 54.4 41.1 58.8 30.3 34.7 16.0 26.5 12.3CR6089 7.3 6.3 60.4 19.4 32.2 7.4 32.8 8.2 CR6241 6.5 4.3 73.5 44.8 48.518.3 38.2 9.6 CR6252 9.8 6.9 74.6 43.6 43.1 25.5 46.5 19.7 CR6388 50.822.6 54.8 18.0 47.2 7.3 51.8 34.1 CR6389 10.5 7.7 56.8 30.8 37.7 19.335.4 16.7 CR6396 6.8 2.9 36.6 9.4 30.9 5.2 37.6 13.1 CR6402 39.0 24.957.9 21.0 36.4 12.9 20.8 6.0 CR6409 46.0 27.6 64.5 36.9 25.0 3.7 46.918.4 CR6415 16.9 12.2 56.6 24.2 35.3 19.6 42.4 20.4 CR6421 5.3 2.9 64.314.0 35.7 21.0 44.9 21.5 CR6429 −0.1 −1.2 58.7 5.7 43.5 12.3 36.5 12.6

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What is claimed is:
 1. A human monoclonal antibody having opsonicphagocytic killing activity against at least one strain of each of atleast two different Enterococcus species and against at least one strainof Staphylococcus aureus, wherein the human monoclonal antibodycomprises: a heavy chain CDR1 comprising SEQ ID NO:326, a heavy chainCDR2 comprising SEQ ID NO:327, a heavy chain CDR3 comprising SEQ IDNO:328, a light chain CDR1 comprising SEQ ID NO:329, a light chain CDR2comprising SEQ ID NO:330, and a light chain CDR3 comprising SEQ IDNO:331.
 2. The human monoclonal antibody of claim 1 wherein the humanmonoclonal antibody comprises: a heavy chain comprising the variableregion of SEQ ID NO: 431, and a light chain comprising the variableregion of SEQ ID NO:
 475. 3. The human monoclonal antibody of claim 1,wherein the at least two different Enterococcus species comprise E.faecalis and E. faecium.
 4. The human monoclonal antibody of claim 2,wherein the at least two different Enterococcus species comprise E.faecalis and E. faecium.
 5. An immunoconjugate comprising: the humanmonoclonal antibody of claim 1, and at least one tag.
 6. A method ofproducing the human monoclonal antibody of claim 1, the methodcomprising: culturing a host cell comprising a vector comprising apolynucleotide encoding said human monoclonal antibody under conditionsconducive to the expression of the human monoclonal antibody.
 7. Themethod according to claim 6, further comprising: recovering theexpressed antibody.
 8. A pharmaceutical composition comprising: thehuman monoclonal antibody of claim 1, and at least one pharmaceuticallyacceptable excipient.
 9. The pharmaceutical composition of claim 8,further comprising: at least one other therapeutic agent.
 10. A methodof producing the human monoclonal antibody of claim 2, the methodcomprising: culturing a host cell comprising a vector comprising apolynucleotide encoding said human monoclonal antibody under conditionsconducive to the expression of the human monoclonal antibody.
 11. Themethod according to claim 10, further comprising: recovering theexpressed antibody.
 12. The human monoclonal antibody of claim 1,wherein the human monoclonal antibody is IgG.
 13. The human monoclonalantibody of claim 12, wherein the IgG is IgG1.